Hets 2021

Anogenital distance in male and female fetuses at 20 to 35 weeks of gestation 947 trimester, (3) a singleton appropriate-for-gestational-age fetus, 4–8 MHz curvilinear abdominal transducer. A pilot study (4) absence of maternal disease, and (5) a normal anatomic has shown us that satisfactory quality imaging of the scanned fetus. Eligibility criterion for inclusion of the data in anogenital region could not be obtained before week 20. the final analysis was the birth of a clinically healthy baby. Measurements were made on the axial plane from 20 weeks of gestation demonstrating the perineal region. First, the Participants were recruited from a low risk, obstetric pop- anus was identified with its circular, hypoechogenic ulation that attended the Chaim Sheba Medical Center Ultra- sphincter, with hyperechogenic mucosa in the center (‘the sound Unit, between 20 and 35 weeks of gestation. The study target sign’), as described by Ochoa et al.14 The distance protocol was approved by the local institutional ethics committee. was measured from the center of the anus to the posterior convergence of the vestibule (fourchette) in female fetuses Sonographic measurements and to the posterior base of the scrotum in male fetuses We used a commercially available Voluson E8 (GE Healthcare, (Figures 1 and 2). Kretztechnik, Zipf, Austria) ultrasound machine, with a Figure 1 An axial plane of the perineum at 31 weeks of gestation, demonstrating measurement in male fetuses Figure 2 An axial plane of the perineum at 31 weeks of gestation, demonstrating measurement in female fetuses Prenatal Diagnosis 2014, 34, 946–951 © 2014 John Wiley & Sons, Ltd. 149

948 Y. Gilboa et al. Sonographic sex assignment was performed according to the Statistics already well known literature.15 Freeze-frame capabilities and The statistical analysis used to calculate the modeled centiles electronic on-screen calipers were used for distance mea- surements. For the first 50 cases and 30 cases, intraobserver and standard deviations (SDs) was based on a previously and interobserver variability was respectively assessed. For described method.16,17 After assessment of normal distribution intraobserver variability, two measurements were carried out, and the mean was calculated. For interobserver variability, using the Kolmogorov–Smirnov test, fitted means and SDs means were calculated from two measurements assessed by two observers (G. Y. and K. Z.). were calculated using the regression models as functions of gestational age, of the raw data (y = a + b*GA + b1*GA2 + b2*GA3), where GA is gestational age. The r2 statistic was also studied to assess the best quality of fit. Figure 3 Intraobserver variability: Bland–Altman plot, mean difference ± 1.96 standard deviation Table 1 Mean anogenital distance (AGD) (mm) by gestational week and sex (raw data) Week of AGD male AGD female Mean difference Mean difference Mean difference gestation fetusesmean ± SD (n) fetusesmean ± SD (n) p (mm + CI 95%) (M/F) % (M/F) ratio 20 9.8 ± 1.4 (11) 7.6 ± 2.1 (10) 0.016 2.2 (0.5–3.9) 28.9 1.3 21 11.8 ± 1.3 (10) 6.9 ± 1.3 (12) <0.001 4.9 (3.7–6.1) 71.0 1.7 22 11.7 ± 1.8 (17) 7.9 ± 1.7 (17) <0.001 3.7 (2.5–5) 48.1 1.5 23 11.8 ± 1.9 (11) 9.3 ± 1.7 (15) 2.5 (1–3.9) 26.9 1.3 24 13.1 ± 1.2 (17) 8.5 ± 1.4 (13) 0.002 4.6 (3.6–5.6) 54.1 1.5 25 14.1 ± 1.8 (12) 9.5 ± 1.7 (14) <0.001 4.7 (3.2–6.1) 48.4 1.5 26 14.1 ± 2.5 (16) 9.3 ± 2.0 (14) <0.001 4.8 (3–6.5) 51.6 1.5 27 14.5 ± 1.9 (12) 9.8 ± 1.6 (13) <0.001 4.7 (3.2–6.1) 48.0 1.5 28 16.3 ± 2.3 (10) 11.6 ± 2.0 (10) <0.001 4.6 (2.5–6.7) 40.5 1.4 29 18.5 ± 1.5 (11) 11.0 ± 1.3 (12) <0.001 7.4 (6.2–8.6) 68.2 1.7 30 17.9 ± 2.0 (10) 12.2 ± 2.3 (10) <0.001 5.8 (3.7–7.8) 46.7 1.5 31 19.8 ± 2.3 (16) 12.6 ± 2.1 (16) <0.001 7.3 (5.6–8.9) 57.1 1.6 32 22.6 ± 2.2 (25) 13.5 ± 1.9 (19) <0.001 9.2 (7.8–10.4) 67.4 1.7 33 22.4 ± 2.3 (17) 14.6 ± 2.3 (10) <0.001 7.9 (5.9–9.8) 53.4 1.5 34 24.4 ± 1.6 (10) 13.3 ± 1.0 (11) <0.001 11.1 (9.8–12.3) 83.5 1.8 35 25.6 ± 2.3 (13) 13.8 ± 2.7 (10) <0.001 11.7 (9.6–13.9) 85.5 1.9 Total 17.0 ± 5.2 (218) 10.7 ± 2.9 (206) <0.001 6.3 (5.5–7.2) 58.9 1.6 <0.001 AGD, anogenital distance; SD, standard deviation; n, number of cases; p, independent Student’s t- test Prenatal Diagnosis 2014, 34, 946–951 © 2014 John Wiley & Sons, Ltd. 150

Anogenital distance in male and female fetuses at 20 to 35 weeks of gestation 949 From the polynomial models for the mean and SD of the interobserver variability was assessed by the interclass variable, the required centiles for a given GA were calculated correlation. For assessing agreement between the two using centilega = meanGA + K SDGA, where K is the corres- measurements, we used the Bland–Altman plot. The mean, ponding centile of the Gaussian distribution (for example, SD, confidence intervals for the mean (95%), and the determination of the 10th and 90th centiles requires significance level for the test that the mean of difference equals K = ± 1.28; determination of the 5th and 95th centiles requires 0 was calculated by the one-sample t-test. K = ± 1.645; and determination of the 2.5th and 97.5th centiles requires K = ± 1.96), and the mean and SD are the predicted Statistical analyses were performed using SPSS statistical values obtained from modeling the original data. The unpaired package version 17 for Windows (SPSS, Inc, Chicago, IL) and Student’s t-test was used to evaluate the difference between Microsoft Excel 2007 software (Microsoft Corp, Richmond, the means of the two sexes. The intraobserver and CA). All tests were two tailed and a p value of <0.05 was considered statistically significant. Table 2 Normal modeled anogenital distance between 20 and 35 weeks of gestation: 2.5th to 97.5th centiles Week 2.5th 5th 10th 25th 50th 75th 90th 95th 97.5th (a) In male fetuses 11.9 12.4 20 5.9 6.4 7.0 8.0 9.2 10.3 11.3 13.1 13.7 21 6.8 7.4 8.0 9.0 10.2 11.4 12.5 14.3 14.9 22 7.7 8.3 9.0 10.1 11.3 12.5 13.6 15.5 16.1 23 8.6 9.2 9.9 11.1 12.4 13.7 14.8 16.7 17.3 24 9.6 10.2 10.9 12.1 13.5 14.8 16.0 17.9 18.6 25 10.5 11.1 11.9 13.1 14.5 15.9 17.2 19.1 19.8 26 11.4 12.1 12.8 14.2 15.6 17.0 18.4 20.3 21.0 27 12.3 13.0 13.8 15.2 16.7 18.2 19.5 21.5 22.3 28 13.2 14.0 14.8 16.2 17.8 19.3 20.7 22.8 23.5 29 14.1 14.9 15.8 17.2 18.8 20.4 21.9 24.0 24.7 30 15.1 15.8 16.7 18.2 19.9 21.6 23.1 25.2 26.0 31 16.0 16.8 17.7 19.3 21.0 22.7 24.2 26.4 27.2 32 16.9 17.7 18.7 20.3 22.1 23.8 25.4 27.6 28.4 33 17.8 18.7 19.7 21.3 23.1 25.0 26.6 28.8 29.7 34 18.7 19.6 20.6 22.3 24.2 26.1 27.8 30.0 30.9 35 19.7 20.6 21.6 23.3 25.3 27.2 29.0 (b) In female fetuses 9.5 10.0 20 4.2 4.7 5.2 6.1 7.1 8.1 9.0 10.1 10.6 21 4.6 5.1 5.7 6.6 7.6 8.6 9.5 10.7 11.1 22 5.1 5.6 6.1 7.1 8.1 9.2 10.1 11.2 11.7 23 5.5 6.0 6.6 7.6 8.6 9.7 10.7 11.8 12.3 24 6.0 6.5 7.1 8.0 9.1 10.2 11.2 12.4 12.9 25 6.4 6.9 7.5 8.5 9.6 10.8 11.8 13.0 13.5 26 6.8 7.4 8.0 9.0 10.2 11.3 12.3 13.5 14.1 27 7.3 7.8 8.4 9.5 10.7 11.8 12.9 14.1 14.7 28 7.7 8.3 8.9 10.0 11.2 12.4 13.5 14.7 15.2 29 8.2 8.7 9.4 10.5 11.7 12.9 14.0 15.2 15.8 30 8.6 9.2 9.8 11.0 12.2 13.5 14.6 15.8 16.4 31 9.0 9.6 10.3 11.5 12.7 14.0 15.1 16.4 17.0 32 9.5 10.1 10.8 11.9 13.2 14.5 15.7 17.0 17.6 33 9.9 10.5 11.2 12.4 13.8 15.1 16.3 17.5 18.2 34 10.4 11.0 11.7 12.9 14.3 15.6 16.8 18.1 18.8 35 10.8 11.4 12.2 13.4 14.8 16.1 17.4 Prenatal Diagnosis 2014, 34, 946–951 © 2014 John Wiley & Sons, Ltd. 151

950 Y. Gilboa et al. RESULTS gradually during pregnancy, with minimal overlapping at over The study included 424 fetuses, 218 male and 206 female, 30 weeks of gestation. This implies a clear-cut differentiation for which adequate measurements could be obtained. In between sexes, only after this period of pregnancy. Our 17 cases, 7 male and 10 female fetuses, all above 32 weeks findings thus confirm the sexually dimorphic characteristics of gestation, adequate measurement could not be obtained of this biometric parameter during fetal life, as has been because of fetal positioning or limiting maternal habitus. recognized in the post natal literature. Interobserver and intraobserver variability measured by the intraclass correlation coefficient was 0.957 (CI 5–95 = Natsuyama18 was the first, and to the best of our knowledge, 0.929–0.974) and 0.899 (CI 5–95 = 0.668–0.994) (r = <0.001), the last, to use the AGD for the assessment of the fetal genitalia. respectively. The mean difference of two measurements A significantly higher mean male to female AGD ratio was by a single observer was 0.122 mm CI 95 = -0.585À0.340 reported for all gestational ages, compared with our findings. p = 0.598 (Figure 3). The mean difference between two Because no details concerning methodology were mentioned, observers was 0.365 mm (CI 95 = -1.154À0.424), p = 0.351 and considering the lower quality of sonographic imaging 30 years ago, the reasons for this difference are elusive. The mean AGD differed significantly between male and female Moreover, the ‘target sign’,14 an important landmark for fetuses at each gestational week. The mean absolute difference accurate measurement of the AGD, according to our increased linearly during gestation from 2.2 mm at 20 weeks of experience, and to that of others,19 is not clearly defined before gestation to 11.8 mm at 35 weeks of gestation (r2 = 0.808). 20 weeks of gestation, even with high resolution ultrasound Accordingly, the ratio of male to female fetuses in mean AGD machines. increased from 1.3 : 1 to 1.9 : 1 (r2 = 0.384) (Table 1). In both sexes, AGD showed a significant linear correlation with gestational age, Two neonatal studies3,12 showed higher values of AGD than r2 = 0.954 and 0.947 for male and female fetuses, respectively. did ours. This difference may be due, at least in part, to the This correlation was expressed by a regression equation: extension of our study until 35 weeks of gestation only, as well for male fetuses, À12.348 + 1.075*GA, and for female fetuses, as to differences due to the racial/ethnic background of the À3.179 + 0.513*GA. The modeled 2.5th to 97.5th centiles, populations. Phillips et al.20 reported that Jewish female between 20 and 35 weeks of gestation, are presented in Table 2a infants had a greater anofourchette distance than did and b. The distribution of individual measurements of fetal AGD Bedouins, and Thankamony A. et al.3 reported that Asian, for both sexes is plotted simultaneously in Figure 4. native Americans, and mixed race infants have smaller AGD than do Caucasian infants. DISCUSSION For the first time in the prenatal literature, we present normal We demonstrated that the AGD measurement is feasible and reference ranges for fetal AGD. We demonstrated significantly reliable. The male to female ratio increased steadily with higher values for male than female fetuses at each week of advancing gestational age to a maximum of 1.9 : 1 respectively gestation. The mean difference between the sexes increased at 35 weeks of gestation. This ratio, considering the expected growing slope, is similar to AGD measurements in term newborn infant studies.3,12 Figure 4 Anogenital distance (AGD): scatterplots of individual measurements, with mean regression curves and 90% reference range of male fetuses (blue dots) and female fetuses (green dots), distributed between 20 and 35 weeks of gestation Prenatal Diagnosis 2014, 34, 946–951 © 2014 John Wiley & Sons, Ltd. 152

Anogenital distance in male and female fetuses at 20 to 35 weeks of gestation 951 Male reproductive disorders are presumed to originate in early WHAT’S ALREADY KNOWN ABOUT THIS TOPIC? fetal life, consequent to abnormality in testicular development.21 • A short anogenital region was reported in babies to be associated Hypospadias, cryptorchidism, testicular feminization, and with cryptorchidism and hypospadias. So far, no information is known regarding the normal distance in male and female fetuses reduced semen production infertility have been associated with throughout gestation. shorter AGD.8,10 Meanwhile, a longer AGD was reported in rats, treated prenatally with testosterone.22 Therefore, our normative WHAT DOES THIS STUDY ADD? • The study shows that anogenital distance measurement in utero is data may add a valuable complementary tool in the assessment of suspected cases of genital anomalies by providing an objective feasible. The measurements assess the normality of the perineal region and may assist in the detection of genital anomalies. parameter during prenatal counseling. Future studies are needed to evaluate AGD measurement in fetuses that develop with abnormal genitalia. REFERENCES 11. Jain VG, Singal AK. Shorter anogenital distance correlates with undescended testis: a detailed genital anthropometric analysis in 1. Michael H. Decrease in anogenital distance among male infants human newborns. Hum Reprod 2013;28:2343–9. with prenatal phthalate exposure. Environ Health Perspect 2005; 113:1056–61. 12. Salazar-Martinez E, Romano-Riquer P, Yanez-Marquez E, et al. Anogenital distance in human male and female newborns: a 2. Torres-Sanchez L, Zepeda M, Cebrian ME, et al. descriptive, cross-sectional study. Environ Health 2004;3:8. Dichlorodiphenyldichloroethylene exposure during the first trimester of pregnancy alters the anal position in male infants. Ann N Y Acad Sci 13. Eisenberg ML, Hsieh TC, Lipshultz LI. The relationship between 2008;1140:155–62. anogenital distance and age. Andrology 2013;1:90–3. 3. Thankamony A, Ong KK, Dunger DB, et al. Anogenital distance from 14. Ochoa JH, Chiesa M, Vildoza RP, et al. Evaluation of the perianal birth to 2 years: a population study. Environ Health Perspect muscular complex in the prenatal diagnosis of anorectal atresia in a 2009;117:1786–90. high-risk population. Ultrasound Obstet Gynecol 2012;39:521–7. 4. Miyata K, Yabushita S, Sukata T, et al. Effects of perinatal exposure to 15. Odeh M, Granin V, Kais M, et al. Sonographic fetal sex determination. flutamide on sex hormones and androgen-dependent organs in F1 male Obstet Gynecol Surv 2009;64:50–7. rats. J Toxicol Sci 2002; 27:19–33. 16. Altman DG, Chitty LS. Charts of fetal size: 1. Methodology. Br J Obstet 5. McIntyre BS, Barlow NJ, Foster PM. Male rats exposed to linuron in Gynaecol 1994;101:29–34. utero exhibit permanent changes in anogenital distance, nipple retention, and epididymal malformations that result in subsequent 17. Royston P, Wright EM. How to construct ‘normal ranges’ for fetal testicular atrophy. Toxicol Sci 2002;65:62–70. variables. Ultrasound Obstet Gynecol 1998;11:30–8. 6. Sathyanarayana S, Beard L, Zhou C, Grady R. Measurement and 18. Natsuyama E. Sonographic determination of fetal sex. Am J Obstet correlates of ano-genital distance in healthy, newborn infants. Int J Gynecol 1984;149:748–57. Androl 2010;33:317–23. 19. Bourdelat D, Muller F, Droullé P, et al. Anatomical and sonographical 7. Gray LE, Ostby J, Furr J, et al. Effects of environmental antiandrogens on studies on the development of fecal continence and sphincter reproductive development in experimental animals. Hum Reprod development in human fetuses. Eur J Pediatr Surg 2001;11:124–30. Update 2001;7:248–64. 20. Phillip M, De Boer C, Pilpel D, et al. Clitoral and penile sizes of full term 8. Hsieh MH, Eisenberg ML, Hittelman AB, et al. Caucasian male infants newborns in two different ethnic groups. J Pediatr Endocrinol Metab and boys with hypospadias exhibit reduced anogenital distance. Hum 1996;9:175–9. Reprod 2012;27:1577–80. 21. Skakkebaek NE, Rajpert-De Meyts E, Main KM. Testicular dysgenesis 9. Paris F, De Ferran K, Bhangoo A, et al. Isolated ‘idiopathic’ micropenis: syndrome: an increasingly common developmental disorder with hidden genetic defects? Int J Androl 2011;34:e518–25. environmental aspects. Hum Reprod 2001;16:972–8. 10. Mendiola J, Stahlhut RW, Jørgensen N, et al. Shorter anogenital distance 22. Wolf CJ, Hotchkiss A, Ostby JS, et al. Effects of prenatal testosterone predicts poorer semen quality in young men in Rochester, New York. propionate on the sexual development of male and female rats: a dose- Environ Health Perspect 2011;119:958–63. response study. Toxicol Sci 2002;65:71–86. Prenatal Diagnosis 2014, 34, 946–951 © 2014 John Wiley & Sons, Ltd. 153



Potential drug interactions in travelers with chronic illnesses: a large retrospective cohort study Travel Medicine and Infectious disease | 2014 ‫ פרופ' אלי שוורץ‬:‫מנחה‬ ‫ דר' שמואל שטיינלוף‬:‫מנחה‬ ‫מכון לרפואה גאוגרפית ומחלות טרופיות‬ '‫מנהל מחלקה פנימית ג‬ [email protected] [email protected] ‫ביאנקה סטרלצין‬ ‫אונ' תל אביב‬ ‫השתתפה כסטודנטית בפרויקט ח״ץ‬ 2010-2012 ‫בין השנים‬ [email protected] 155

Travel Medicine and Infectious Disease (2014) 12, 499e504 Available online at www.sciencedirect.com ScienceDirect journal homepage: www.elsevierhealth.com/journals/tmid Potential drug interactions in travelers with chronic illnesses: A large retrospective cohort study* Shmuel Stienlauf a,b,c,*, Eyal Meltzer a,b,c, Daniel Kurnik b,c, Eyal Leshem a,b,c, Eran Kopel a,b, Bianca Streltsin c,d, Eli Schwartz a,b,c a The Center of Geographic Medicine, The Chaim Sheba Medical Center, Tel Hashomer 52621, Israel b The Departments of Internal Medicine, The Chaim Sheba Medical Center, Tel Hashomer 52621, Israel c The Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel d The Arrow Project, The Chaim Sheba Medical Center, Tel Hashomer, 52621, Israel Received 28 February 2014; received in revised form 9 April 2014; accepted 24 April 2014 Available online 5 May 2014 KEYWORDS Summary Background: Data regarding the prevalence of potential interactions between travel-related medications (TRM) and chronic medications in use, or medical conditions of Travelers; travelers to developing countries are limited. Chronic disease; Methods: A retrospective cohort study of travelers to low income countries. We extracted data on Chronic use of demographics, travel destinations, use of chronic medications, drug allergies, and relevant med- prescription drugs; ical conditions. The following TRM were evaluated: mefloquine, primaquine, doxycycline, atova- Israel; quone/proguanil, fluoroquinolone antibiotics, rifaximin, azithromycin, and acetazolamide. Developing countries; Results: A total of 16,263 travelers were included in the analysis, of whom 3299(20%) suffered Drug interactions from chronic illnesses and 2316(14%) reported chronic medication use. A potential drugedrug interaction with TRM was identified in 1047(45%) of travelers using chronic medication. Fluoroquinolones and azithromycin were the most commonly implicated TRMs. A potential medical condition interaction with TRM was identified in 717(22%) of travelers hav- ing chronic illnesses. acetazolamide, primaquine and mefloquine, were the most commonly TRMs implicated. * Parts of the work were presented at the: (a.) Annual Meeting of the Israel Society for Parasitology, Protozoology and Tropical Diseases, Ramat Gan, Israel, December 12, 2012. (b.) 13th Conference of the International Society of Travel Medicine, May 19e23, 2013, Maastricht, The Netherlands. * Corresponding author. The Department of Internal Medicine “C”, Sheba Medical Center, Tel Hashomer 52621, Israel. Tel.: þ972 52 6666131; fax: þ972 3 5535953. E-mail addresses: [email protected], [email protected] (S. Stienlauf), [email protected] (E. Meltzer), d_ [email protected] (D. Kurnik), [email protected] (E. Leshem), [email protected] (E. Kopel), [email protected] (B. Streltsin), [email protected] (E. Schwartz). http://dx.doi.org/10.1016/j.tmaid.2014.04.008 1477-8939/ª 2014 Elsevier Ltd. All rights reserved. Downloaded for Anonymous User (n/a) at Sheba Medical Center from ClinicalKey.com by Elsevier on October 19, 2021. For personal use only. No other uses without permission. Copyright ©2021. Elsevier Inc. All rights reserved. 156

500 S. Stienlauf et al. Drug allergies, which can pose a relative contraindication for use of acetazolamide, were reported by 1323(8.1%) of all travelers. Conclusions: Potential drugedrug and drug-disease interactions involving TRM might occur in a significant proportion of travelers with chronic medical conditions. Education of health practi- tioners regarding such potential drug interactions and caution when in prescribing travel- related medications is warranted. ª 2014 Elsevier Ltd. All rights reserved. Introduction for chronic medical conditions, and healthy women who used oral contraceptives. Travelers to developing countries who consult a travel clinic prior to their departure are commonly prescribed travel Drug interactions with travel related medications: We related medications (TRM) for malaria prophylaxis, stand- defined the following medications as travel related medi- by therapy for traveler’s diarrhea, or for prevention/ cations (TRM): Drugs used for malaria prophylaxis (meflo- treatment of high altitude sickness. Between 11 and 59% of quine, primaquine, doxycycline and atovaquone/proguanil travelers to developing countries, who consulted a pre- [Malaroneª]), medications prescribed as stand-by therapy travel clinic, report having at least 1 chronic medical con- for traveler’s diarrhea (fluoroquinolone antibiotics [cipro- dition, most of whom were regularly taking prescription floxacin, ofloxacin], rifaximin and azithromycin), and the medications [1e4]. There are potential drugedrug in- carbonic anhydrase inhibitor acetazolamide, used for the teractions between TRM and many other medications, and prophylaxis and therapy of high altitude sickness. caution is advised for their concomitant use [5]. Further- more, medical conditions and drug allergies may pose a risk We used Micromedex� Healthcare Series database [5] to for the use of several travel related medications [6,7]. extract all medications considered to have interactions with TRM, including the effect of each interaction, its Data regarding the prevalence of possible interactions severity (mild, moderate, major, Contraindicated), and the between travel-related medications and chronic medical quality of documentation in the medical literature (Excel- conditions and medications used by travelers to developing lent: Controlled studies have clearly established the exis- countries are lacking. Our objective was to assess the tence of the interaction; Good: Documentation strongly prevalence and types of potential drugedrug and suggests the interaction exists, but well-controlled studies drugedisease interactions among a cohort of travelers to are lacking; Fair: Available documentation is poor, but developing countries attending our pre-travel clinic. pharmacologic considerations lead clinicians to suspect the interaction exists; or, documentation is good for a phar- Materials and methods macologically similar drug). We performed a cohort study based on the medical records The Micromedex database classifies the severity of the of all travelers attending the pre-travel clinic at the Chaim drugedrug interaction as follows [5]: Contraindicated: The Sheba Medical Center between January 1st, 2005 and drugs are contraindicated for concurrent use; Major e The December 31st, 2007. Travelers to developed countries [8] interaction may be life-threatening and/or require medical or those with missing data about travel destination were intervention to minimize or prevent serious adverse ef- excluded. The study was approved by the Sheba Medical fects; Moderate e The interaction may result in exacerba- Center Institutional Review Board. tion of the patient’s condition and/or require an alteration in therapy; Minor e The interaction would have limited The Sheba Medical Center pre-travel clinic provides clinical effects, e.g. increase the frequency or severity of medical advice to approximately 6000e7000 travelers the side effects, but generally would not require a major annually. At the clinic visit, every traveler completes a alteration in therapy. structured questionnaire. The data collected includes age and gender, travel destinations, planned duration of travel, Chronic illness and TRM interactions: We used Micro- expected date of travel, and purpose of travel. Travelers medex� Healthcare Series database [5] to extract all are asked to select one of the following purposes for their medical conditions considered to have interactions with trip: leisure/tourism, business, governmental/non- TRM and its severity (Precaution needed, Contraindicated). governmental organization worker, research/education, returning to region of origin of self or family to visit friends Statistical analysis: Continuous data were described as and relatives (VFR). Travelers are also asked to record mean Æ standard deviation, or median with interquartile chronic medical conditions, chronic use of medication, and range (IQR). Proportions and 95% confidence intervals were drug allergies. All data are stored in a computerized data- calculated for categorical variables using the Wilson base using Microsoft Access� 2010 (Microsoft, Redmond, method [9]. WA, USA), which served as the source for the present study. Funding: No funding source was provided for the design Chronic use of medications was based on self-reporting. conduct and reporting of the study. We analyzed separately, travelers who used medications Results During the study period, 16,263 travelers to developing countries sought pre-travel advice and were included in the Downloaded for Anonymous User (n/a) at Sheba Medical Center from ClinicalKey.com by Elsevier on October 19, 2021. For personal use only. No other uses without permission. Copyright ©2021. Elsevier Inc. All rights reserved. 157

Potential drug interactions in travelers with chronic illnesses 501 Table 1 Demographics and travel destinations. The severities of the interactions were rated as contra- indicated, major, moderate and minor in 4(0.4% of all trav- Parameter All Travelers on Travelers not elers with interactions), 684(65%), 288(28%) and 885(48%) travelers travelers respectively. The quality of the documentation of chronic using chronic the drugedrug interaction was rated excellent, good, or fair, in 10%, 61% and 29% of the interactions, respectively. medications medications Table 2 lists the prevalence of potential drugedrug Number, n (%) 16,263 2316 (14) 13,947 (86) interaction involving TRMs. Table 3 lists the common chronic Females, n (%) 7859 (48) 1168 (50) 6691 (48) medications that have interactions with TRM, and the po- Age tential detrimental effect of that drugedrug interaction. Mean (ÆSD) 32.2 Æ 14.5 47.6 Æ 17 29.7 Æ 12.3 Median (IQR) 26 (22e40) 53 (29e53) 25 (22e32) Table 4 summarizes the interactions between TRM and Destination: 10,620 (65) 1467 (63) 9153 (66) chronic medical conditions. Of 3299 travelers with chronic medical conditions, 717(22%, 95% CI, 20%e23%) were at risk Asia 4023 (25) 533 (23) 3490 (25) for potential interactions with TRM. Acetazolamide (240, Latin America 1896 (12) 393 (17) 1503 (11) 29% of travelers with TRM-chronic illness interactions), Sub-Saharan followed by primaquine (236, 29%) and mefloquine (191, 23%) were the most common TRM implicated. Africa Drug allergies, which can pose a relative contraindica- study. Demographics and travel destinations are reported in tion for use of acetazolamide, were reported by 1323 (8.1%) Table 1. of all travelers, including 137 (0.8%) who reported allergy to sulfonamides. Oral contraceptive usage was reported by 817 (14% of female travelers <40 years). With the exception of doxy- The most frequent prescribed medications with a po- cycline, which can lead to a decrease in contraceptive tential of drugedrug interaction with TRMs were the effectiveness, no other TRM oral contraceptives in- cholesterol lowering drugs (simvastatin (n Z 417; 13% of teractions were found. chronic medication users), atorvastatin (n Z 123, 4%), followed by the anti-diabetic drug metformin (n Z 104; Out of the 16,263 travelers, 3299(20%) reported having 3%), (Table 3). chronic illnesses. Chronic medication usage, other than oral contraceptives was reported by 2316 travelers (14%), one or Approximately 30% of travelers on chronic medications more of 377 different prescription medications, with mean were at risk for potential drugedrug interactions with an- number of medications of 1.8 Æ 1.3 per patient tibiotics for traveler’s diarrhea, making it the TRM class (median Z 1 [1,2]; range, 1e12). with the largest number of potential drugedrug in- teractions. In addition, the use of fluoroquinolones was not Chronic medications included, in descending order, advised in 0.3% (n Z 51) of all travelers due to central medications used for cardiovascular diseases (46%), lipid nervous system diseases, which may increase the risk of lowering agents (31%), hormones, mainly thyroid prepara- seizures. Rifaximin - a non-absorbable antimicrobial, had tions (20%), anti-agregants/anticoagulants (14%), medica- the lowest potential for drug interactions, its sole inter- tions for respiratory diseases (16%), medications for action was with warfarin, resulting in decreased anticoag- gastrointestinal disorders (10%), psychotherapeutic agents ulant efficacy of warfarin. (10%), and medications for diabetes (10%). Drugs used for malaria prophylaxis, were the second Potential drugedrug interactions: The Micromedex most frequent cause of drugedrug interactions with TRMs, database [5] listed potential drugedrug interactions with affecting approximately 12% of travelers on chronic medi- 66 different medications (18% of 377 prescription medica- cations. Mefloquine had the greatest potential for druge- tions used by the travelers) used among the travelers. Of drug interactions, mainly with selective serotonin re- 2316 travelers on chronic medications, 1047 (45%; 95% CI, uptake inhibitors (Tables 2 and 3). In addition, mefloquine 43%e47%) travelers were considered at risk for potential drug interactions with TRM. Table 2 Travel-related medications and potential drugedrug interactions with chronic medications. Travel-related medications No. of travelers on chronic No. of travelers with Total number of interactionsb medications with potential potential major/moderate interactions (%)a interactions (%)a 817 684 Fluoroquinolone 707 (23) 618 (20) 167 Azithromycin 654 (21) 519 (17) 152 Mefloquine 161 (7) 135 (6) 123 Doxycycline 148 (6) 139 (6) Acetazolamide 122 (4) 106 (3) 24 Atovaquone/proguanil 22 Rifaximin 24 (1) 22 (1) Primaquine 22 (1) 0 (0) 0 0 (0) 0 (0) a % of travelers using chronic medications. b Travelers may take more than 1 drug with potential interaction. Downloaded for Anonymous User (n/a) at Sheba Medical Center from ClinicalKey.com by Elsevier on October 19, 2021. For personal use only. No other uses without permission. Copyright ©2021. Elsevier Inc. All rights reserved. 158

502 S. Stienlauf et al. Table 3 Common drug interactions between travel-related and chronic medications. Travel related Interacting Travelers Severity Documentation Interaction effect n (%)a medication drug Fluoro- Simvastatin 417 (13) Major Good Myopathy or rhabdomyolysis. quinolones Metformin 104 (3) Major Excellent Increased risk of hypoglycemia or hyperglycemia. Salmeterol Major Fair Increased risk of QT interval prolongation. Azithromycin Metoprolol 40 (1) Minor Good Bradycardia, hypotension. Insulin 35 (1) Major Excellent Increased risk of hypoglycemia or hyperglycemia. Citalopram 32 (1) Major Fair Increased risk of QT interval prolongation. Simvastatin 30 (1) Major Good Rhabdomyolysis. Atorvastatin 417 (13) Moderate Good Rhabdomyolysis. Salmeterol 123 (4) Major Fair QT interval prolongation. Citalopram 40 (1) Major Fair Increased citalopram exposure and increased 30 (1) risk of QT interval prolongation. Increased risk of bleeding. Mefloquine Warfarin 22 (1) Major Good Increased serum carbamazepine levels. Carbamazepine 16 (1) Minor Fair Increased risk of QT interval prolongation. Salmeterol 40 (1) Major Fair Increased risk of QT interval prolongation. Citalopram 30 (1) Major Fair Increased risk of cardiotoxicity (QT prolongation, Fluoxetine 17 (1) Major Fair torsades de pointes, cardiac arrest). Loss of seizure control Carbamazepine 16 (1) Moderate Fair Increased risk of ECG abnormalities and cardiac arrest. Propranolol 15 (1) Major Good Decreased effectiveness of doxycycline. Doxycycline Calcium 62 (3) Moderate Good An increased risk of bleeding. preparations Pseudotumor cerebri (benign intracranial Warfarin 22 (1) Moderate Fair hypertension). Isotretinoin 19 (1) Decreased doxycycline effectiveness. Major Good An increased risk of methotrexate toxicity (leukopenia, thrombocytopenia, anemia, Carbamazepine 16 (1) Moderate Fair nephrotoxicity, mucosal ulcerations). Lactic acidosis. Methotrexate 13 (1) Major Good Acetazolamide Metformin 104 (3) Major Fair a % of travelers who are taking medications regularly. Table 4 Types & severity of travel related medications and chronic medical conditions interactions. Travel related medication Chronic medical condition Interaction severity n(%)a n(%)b Acetazolamide Diabetes mellitus Precaution needed 212(7.3) 240(7.3) Liver diseases Precaution needed 8(0.24) Primaquine COPD Precaution needed 3(0.09) 236(7.15) Mefloquine Addison’s syndrome Contraindicated 1(0.03) 227(6.9) Fluoroquinolone antibiotics Myasthenia gravis Precaution needed 2(0.0.6) Atovaquone/proguanil Cirrhosis Contraindicated 0 68(2.1) Rifaximin Renal Failure Contraindicated 0 0 Azithromycin G6PD deficiency Contraindicated 236(7.15) Psychiatric disease Contraindicated 182(5.52) Epilepsy Precaution needed 34(1.03) Liver diseases Precaution needed 9(0.27) CNS disorders Precaution needed 66(2.0) Myasthenia gravis Precaution needed 2(0.06) Renal failure Precaution needed 0 Renal failure Contraindicated 0 None reported None reported a n- Number of travelers with potential TRM-chronic disease interaction, % of travelers with chronic illnesses. b n- Number of all travelers with the TRM who have potential interactions with chronic diseases interaction, % of travelers with chronic illnesses. Downloaded for Anonymous User (n/a) at Sheba Medical Center from ClinicalKey.com by Elsevier on October 19, 2021. For personal use only. No other uses without permission. Copyright ©2021. Elsevier Inc. All rights reserved. 159

Potential drug interactions in travelers with chronic illnesses 503 use was contraindicated due to neuropsychiatric diseases in diseases and fluoroquinolones) should be carefully evalu- 219 (1.3%) of all travelers. The most common drugedrug ated before prescribing fluoroquinolones or azithromycin interaction for doxycycline was with calcium supplemen- for the self-treatment of traveler’s diarrhea. tation medication, resulting potentially in decreased doxycycline effectiveness. The most common drugedrug Travel to high altitude areas is very common [25]. The interaction for atovaquone/proguanil was with warfarin, main health hazard of high altitudes is the various forms of potentially resulting in an enhanced anticoagulant effect. high altitude illness, that can be prevented by the pro- Primaquine did not have any drugedrug interactions with phylactic use of acetazolamide [26,27]. Caution should be medications used by our travelers, but was contraindicated practiced in the prescription of altitude sickness prophy- in 236 travelers (1.5% of all travelers) who reported laxis. Concomitant therapy of acetazolamide and metfor- glucose-6-phosphate dehydrogenase deficiency (G6PD). min (used by 3% of travelers on chronic medications in our cohort) is associated with increased risk for lactic acidosis Acetazolamide had one major interaction- with met- [5]. Acetazolamide may impair blood glucose control in formin, which was reported in 3% of travelers who were on diabetic patients [5], and should be used with caution in chronic medications (Table 3). Moreover, caution was diabetic patients. Another source of concern is the risk of advised using acetazolamide in all diabetic patients (1.3% induction of ketosis by acetazolamide in patients with type of all travelers), due to the potential for glycemic control 1 diabetes [28e30]. Acetazolamide was associated with worsening and risk for acid/base and electrolyte imbal- ketonuria in 4 out of 15 diabetic climbers, two of them ances. Drug allergies, which can pose a relative contrain- required treatment for ketoacidosis [29]. dication for use of acetazolamide, were reported by 1323 (8.1%) of all travelers, including 137 (0.8%) who reported Thus, currently available data suggests that the admin- allergy to sulfonamides. istration of acetazolamide for the prevention of high- altitude sickness should be avoided in diabetic patients, Discussion particularly if they are on metformin therapy. In our cohort, almost half (45%) of travelers on chronic Malaria remains the most important infectious risk for medications were using drugs with potentially harmful travelers to many developing countries. Yet, current drugedrug interactions with TRMs, 85% of which were of guidelines and treatment recommendations fail to consider major or moderate severity. Among TRMs, the antibiotics the traveler’s chronic medication use [31e34]. Atovaquone- fluoroquinolones and azithromycin have the highest poten- proguanil, mefloquine, and doxycycline can be used for tial for drugedrug interaction with chronic medications. malaria prophylaxis, including in areas with chloroquine- Statins and metformin were the most common chronic resistant malaria, while primaquine can be also used for medications implicated in drugedrug interactions with TRM. prophylaxis against Plasmodium vivax malaria [31] In our study, mefloquine had potential for severe drugedrug in- About one fifth (22%) of travelers with chronic illnesses. teractions (QT prolongation and cardiac arrhythmias) in 6% had the potential for disease-TRM interaction, mainly with of travelers on chronic medications (Table 3) and was acetazolamide, primaquine and mefloquine. contraindicated in almost 1% of travelers, those who had neuro- psychiatric diseases. Doxycycline had the potential How should these data affect advice to travelers? Some to cause significant drug interaction in 6% of travelers on of the main treatment paradigms concerning travel related chronic medications. In addition, conflicting data exist in illness, such as traveler’s diarrhea, were established many the literature as to whether doxycycline reduces the effi- years ago, in an era when travel to developing countries cacy of oral contraceptives [5,35,36]. In contrast, was dominated by backpackers e healthy young adults. In atovaquone-proguanil and primaquine had fewer potential this population, enthusiasm for antimicrobial therapy drugedrug interactions. Primaquine is contraindicated in hinges on the perception that the small likelihood of harm subjects with G6PD deficiency, which was present in at from therapy is far outweighed by the possibility of benefit. least 1.2% of our cohort. Similarly, atovaquone-proguanil However, traveler’s diarrhea is rarely associated with se- has potential interactions with antiretroviral agents, vere morbidity, let alone any mortality [10e16]. necessitating caution in travelers treated for HIV. In our study, up to 30% of travelers on chronic medica- In contrast to the use of TRM in travelers’ diarrhea or for tions had potential moderate to severe drugedrug in- high altitude sickness, which usually are for short duration, teractions with antibiotics for the self-treatment of malaria prophylaxis is of a long-term use which increases traveler’s diarrhea, i.e., fluoroquinolone antibiotics or the potential for drugedrug or drug-disease interactions. azithromycin. The main potential adverse effects were rhabdomyolysis and QT interval prolongation, resulting in Our study has several limitations. It is a single center potentially fatal cardiac arrhythmias. Most of the recom- study performed in a single country. Generalization of our mendations for self-treatment of traveler’s diarrhea are finding can be hampered by different patterns of chronic based on studies that were performed in healthy young medication usage and TRM prescription between countries adults. Travelers with “significant underlying medical and travel clinics. It should be noted however, that there is problems” were excluded [17e24]. To date, most reviews a similarity in the use of chronic medications by travelers in and guidelines regarding traveler’s diarrhea have failed to USA [2], France [4] and Israel (present study). Referral bias acknowledge the potential risks of TRMs in diverse pop- may also exist, as it is not known whether prescription drug ulations. Our findings suggest that in travelers with chronic users are more or less likely to be referred, or refer diseases and chronic medication use, potential drugedrug themselves to pre-travel consultation. and drugedisease interactions (i.e. central nervous system In conclusion, in travelers taking chronic medications, there is a high potential for drugedrug and drug-disease interactions with TRMs. Healthcare practitioners need to be Downloaded for Anonymous User (n/a) at Sheba Medical Center from ClinicalKey.com by Elsevier on October 19, 2021. For personal use only. No other uses without permission. Copyright ©2021. Elsevier Inc. All rights reserved. 160

504 S. Stienlauf et al. aware of theses hazards and have to balance the expected comparable to levofloxacin for the treatment of US travelers benefits and potential risks of TRMs when consulting trav- with acute diarrhea acquired in Mexico. Clin Infect Dis 2003;37: elers to developing countries. 1165e71. [18] DuPont HL, Ericsson CD, Mathewson JJ, DuPont MW. Five Conflict of interest statement versus three days of ofloxacin therapy for traveler’s diarrhea: a placebo-controlled study. Antimicrob Agents Chemother None declared. 1992;36:87e91. [19] DuPont HL, Jiang ZD, Ericsson CD, Adachi JA, Mathewson JJ, Acknowledgments DuPont MW, et al. Rifaximin versus ciprofloxacin for the treatment of traveler’s diarrhea: a randomized, double-blind No funding source was available for this study. clinical trial. Clin Infect Dis 2001;33:1807e15. [20] Flores J, DuPont HL, Jiang Z-D, Okhuysen PC, Melendez- References Romero JH, Gonzalez-Estrada A, et al. 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[13] Hill DR, Beeching NJ. Travelers’ diarrhea. Curr Opin Infect Dis [36] Tan KR, Magill AJ, Parise ME, Arguin PM. Doxycycline for ma- 2010;23:481e7. laria Chemoprophylaxis and treatment: report from the CDC expert meeting on malaria chemoprophylaxis. Am J Trop Med [14] Shlim DR. Treatment of travelers’ diarrhea. In: Keystone JS, Hyg 2011;84:517e31. Kozarsky PE, Freedman DO, Nothdurft HD, Connor BA, editors. Travel medicine. 2nd ed. Elsevier Inc; 2008. pp. 205e9. [15] Soonawala D, Vlot JA, Visser LG. Inconvenience due to trav- elers’ diarrhea: a prospective follow-up study. BMC Infect Dis 2011;11:322. [16] Steffen R. Epidemiology of traveler’s diarrhea. Clin Infect Dis 2005;41:S536e40. [17] Adachi JA, Ericsson CD, Jiang ZD, DuPont MW, Martinez- Sandoval F, Knirsch C, et al. Azithromycin found to be Downloaded for Anonymous User (n/a) at Sheba Medical Center from ClinicalKey.com by Elsevier on October 19, 2021. For personal use only. No other uses without permission. Copyright ©2021. 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Low molecular weight heparin therapy during pregnancy is associated with elevated circulatory levels of placental growth factor Placenta | 2014 ‫ פרופ' שלמה ליפיץ‬:‫מנחה‬ ‫ פרופ' יואב ינון‬:‫מנחה‬ ‫אולטרסאונד גינקולוגי‬ ‫אולטרסאונד גינקולוגי‬ [email protected] [email protected] ‫אלעד בן מאיר‬ ‫אונ' תל אביב‬ ‫השתתף כסטודנט בפרויקט ח“ץ‬ 2011-2012 ‫בין השנים‬ [email protected] 163

Placenta 36 (2015) 121e124 Contents lists available at ScienceDirect Placenta journal homepage: www.elsevier.com/locate/placenta Low molecular weight heparin therapy during pregnancy is associated with elevated circulatory levels of placental growth factor Y. Yinon*, 1, E. Ben Meir 1, L. Margolis, S. Lipitz, E. Schiff, S. Mazaki-Tovi, M.J. Simchen Department of Obstetrics and Gynecology, Sheba Medical Center, Tel-Hashomer, Sackler School of Medicine, Tel-Aviv University, Israel article info abstract Article history: Introduction: Low molecular weight heparin (LMWH) has been shown to be effective in decreasing the Accepted 12 December 2014 recurrence of placenta-mediated complications of pregnant women. The aim of this study was to determine the effect of LMWH on circulating levels of soluble fms-like tyrosine kinase-1 (sFlt-1), soluble Keywords: endoglin (sEng) and placental growth factor (PLGF) in pregnant women who required anticoagulation Low molecular weight heparin therapy. Angiogenic factors Methods: A longitudinal prospective cohort study was performed including pregnant women in whom sFlt-1 anticoagulation therapy by LMWH during pregnancy was clinically indicated (n ¼ 33). Healthy pregnant PLGF women, matched for gestational age, who did not require thromboprophylaxis served as controls sEng (n ¼ 29). Maternal plasma samples were obtained throughout gestation every 4 weeks and stored at �70 �C. Maternal plasma concentrations of sFlt-1, sEng and PLGF were determined by ELISA and compared between the two groups. Results: Patients treated with LMWH had significantly increased circulatory levels of PLGF during the third trimester compared with controls (28e34 weeks: 719.2 pg/ml vs 558.6 pg/ml at, p < 0.01; 35e40 weeks: 975.6 pg/ml vs 511.2 pg/ml, p < 0.01, respectively). In contrast, circulatory levels of sFlt-1 and sEng were similar between the LMWH treatment group and controls throughout gestation. Consistent with these findings, the ratio of sFlt-1/PLGF was lower in patients treated with LMWH compared to controls (28e34 weeks: 1.9 vs 7.2, p < 0.05; 35e40 weeks: 5 vs 12.9, p < 0.05, respectively). Discussion: Anticoagulation treatment of pregnant women with LMWH is associated with a pro- angiogenic state. These findings may explain the effectiveness of LMWH in the prevention of placenta-mediated complications of pregnancy. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction b receptor Endoglin, are increased in preeclampsia and are associ- Preeclampsia is a clinical syndrome defined as the new onset of ated with a marked increase in the circulatory levels of these hypertension and proteinuria during the second half of pregnancy peptides [4e7]. sFlt-1 antagonizes the pro-angiogenic factors [1] affecting 3e5% of pregnant women worldwide and is a major vascular endothelia growth factor (VEGF) and placental growth cause of maternal and neonatal morbidity and mortality [2,3]. factor (PLGF) by binding them in the circulation thus preventing Recent studies suggest that imbalance of endogenous angiogenic interaction with their endogenous receptors. Notably, clinical factors plays a key role in the pathogenesis of preeclampsia. studies have confirmed that the increase in maternal circulating Placental expression of anti-angiogenic factors soluble fms-like sFlt-1 precedes the clinical manifestation of preeclampsia by 5e6 tyrosine kinase- 1 (sFlt-1), a soluble splice variant of the VEGF- weeks and is correlated with disease severity [4,5,8,9]. Moreover, receptor Flt-1, and soluble endoglin (sEng), a soluble form of TGF- circulatory PLGF levels are decreased in preeclampsia as early as the first trimester, before the sFlt-1 rise, suggesting that an imbalance * Corresponding author. Department of Obstetrics and Gynecology, Sheba Med- of antiangiogenic and proangiogenic factors rather than the level of ical Center, Tel-Hashomer, 52621, Israel. Tel.: þ972 54 6744141; fax: þ972 3 either sFlt-1 or PLGF alone plays a role in the pathophysiology of 5303168. preeclampsia [5,10]. These findings, and others, have led to the notion that treatment strategies aiming at restoring the normal E-mail address: [email protected] (Y. Yinon). angiogenic balance in the maternal circulation could potentially 1 These authors contributed equally to this work. prevent or attenuate the preeclamptic phenotype [11]. http://dx.doi.org/10.1016/j.placenta.2014.12.008 0143-4004/© 2014 Elsevier Ltd. All rights reserved. Downloaded for Anonymous User (n/a) at CONSORTIUM MEDICAL LIBRARIES - ISRAEL -Chaim Sheba Medical Center from ClinicalKey.com by Elsevier on May 12, 2018. For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. 164

122 Y. Yinon et al. / Placenta 36 (2015) 121e124 Since thrombosis in the uteroplacental circulation is frequently conducted using the IBM Statistical Package for the Social Sciences (IBM SPSS v.19; observed in placental-mediated complications of pregnancy, IBM Corporation Inc, Armonk, NY, USA). including preeclampsia, intra-uterine growth restriction (IUGR), placental abruption and intra-uterine fetal death (IUFD), anti- 3. Results coagulation seemed to be a promising therapeutic option [12]. However, Kupferminc et al. have shown that 50% of women treated Demographic and clinical characteristics of the patients are by low molecular weight heparin (LMWH) had placental infarcts, shown in Table 1. Maternal age, rate of primigravidity and pre- which was similar to their control arm [13]. In addition, Rey et al. pregnancy BMI were similar between the two groups. The rate of reported that LMWH was effective in decreasing the recurrence of preeclampsia as well as IUGR did not differ between the two placental-mediated complications even in women without a groups. As expected, in patients treated by LMWH there was a trend known thrombophilia [14]. These findings suggest that LMWH may towards earlier delivery (38.3 vs 38.8 weeks, p ¼ 0.053) and exert potentially beneficial effects on placental function via other, consequently their neonates were smaller than controls (2805 vs non anti-thrombotic pathways. 3182 g, p ¼ 0.037). For the purpose of analysis of the data, we grouped obtained blood samples into three pregnancy/time cate- Heparin induces cytotrophoblast proliferation and attenuates gories: second trimester (16e27 weeks of gestation), early third trophoblast apoptosis [15,16]. Recently, in-vitro studies have trimester (28e34 weeks) and late third trimester (35e40 weeks of showed that LMWH stimulated sFlt-1 release from placental villous gestation). The samples were grouped in this manner based on explants [17,18]. Consistent with the in-vitro data, heparin treat- previous data showing that in normal pregnancies sFlt-1 concen- ment was associated with increased circulatory levels of sFlt-1 [18]. trations remain constant until 34 weeks of gestation and then in- This paradox between heparin's protective effect and its up regu- crease until delivery, while PLGF levels peak at 28 weeks of lation of circulating sFl-1 has not been settled. gestation, and decrease after 34 weeks [5]. The plasma levels of sFlt-1 were similar between study group and controls throughout In the present study, we investigated the effect of LMWH on gestation (Table 2). In contrast, LMWH treatment was associated circulating levels of sFlt-1, sEng and PLGF in pregnant women who with significantly increased circulatory levels of PLGF in the early required anticoagulation therapy. Serial measurements of plasma third trimester starting at 28 weeks of gestation (p < 0.01, Fig. 1 and levels of sFlt-1, sEng and PLGF were performed throughout preg- Table 2). Similarly, circulatory levels of PLGF were higher in patients nancy in LMWH treated women, as well as in pregnant women who receiving LMWH compared with controls in the late third trimester did not require thromboprophylaxis. (p < 0.01, Fig. 1 and Table 2). During the second trimester serum levels of PLGF did not differ between the two groups (p ¼ 0.9, Fig. 1 2. Materials and methods and Table 2). The increased circulatory PLGF levels in patients treated with LMWH without significant change in sFlt-1 levels This was a prospective cohort study of patients in whom anticoagulation ther- resulted in decreased sFlt-1/PLGF ratio in that group compared to apy with LMWH during pregnancy was clinically indicated. Patients were enrolled controls both at 28e34 weeks of gestation and at 35e40 weeks of between June 2012 and December 2013 at a single tertiary center. The study was gestation (Fig. 2 and Table 2, p < 0.05). LMWH treatment was not approved by the local institutional ethics committee, and all patients provided associated with alterations in circulatory levels of sEng throughout written informed consent. The study group included 33 patients who were treated gestation (Table 2). No difference in circulatory PLGF levels was by LMWH throughout gestation and were matched by gestational age to 29 healthy found between the 5 patients who received treatment dose of pregnant women with uncomplicated pregnancies who did not require thrombo- LMWH and the patients who received prophylactic dose. Likewise, prophylaxis and served as controls. The indications for LMWH administration in the there were no differences in circulatory levels of these peptides study group included previous thromboembolism and thrombophilia (n ¼ 11), bad between patients who were treated with aspirin and patients who obstetric history and thrombophilia (n ¼ 7), antiphospholipid syndrome (n ¼ 5), were not and between patients with (n ¼ 27) or without throm- inherited thrombophilia without previous thromboembolism or bad obstetric his- bophilia (n ¼ 6) who received LMWH. tory (n ¼ 4), previous thromboembolism without thrombophilia (n ¼ 4), and bad obstetric history without thrombophilia (n ¼ 2). Bad obstetric history was defined as 4. Discussion previous early-onset preeclampsia or IUGR requiring delivery prior to 34 weeks of gestation, placental abruption, IUFD or preterm birth less than 34 weeks. All patients The evidence that LMWH treatment may promote improved were already undergoing LMWH treatment upon recruitment to the study. Most perinatal outcomes [14,20,21] has prompted us to investigate its patients (28 of 33) received prophylactic dose of 1 mg enoxaparin per kg body effect on circulating angiogenic and anti-angiogenic factors during weight daily, while 5 patients with previous thromboembolism received a treatment pregnancy, as these play a key role in the pathogenesis of pre- dose of 1 mg enoxaparin per kg body weight twice daily. All patients with anti- eclampsia. Herein, we demonstrated for the first time that LMWH phospholipid syndrome (n ¼ 9) were also treated by low dose aspirin (100 mg/d). treatment was associated with increased circulating levels of PLGF Patients with chronic hypertension, pre-gestational diabetes, chronic renal disease, during the third trimester without alteration in the circulating as well smokers, were excluded. Gestational age was determined based on men- strual history and first trimester ultrasound. Demographic and clinical data, ultra- Table 1 sound findings and perinatal outcomes were entered prospectively into a Demographic and clinical characteristics. computerized database. Preeclampsia was defined as blood pressure �140/ 90 mmHg measured on two occasions at least 4 h apart, accompanied by proteinuria Maternal age (years) LMWH (n ¼ 33) Control (N ¼ 29) P (�300 mg/24 h or 2 þ dipstick) occurring after 20 weeks of gestation in a previously Primigravida (%) normotensive woman [19]. Intra-uterine growth restriction (IUGR) was defined as Pre-pregnancy BMI (kg/m2) 33 (30.5e35.5) 32.5 (29e34) 0.94 birthweight below the 5th percentile. Preeclampsia (%) 30.3 28 0.8 IUGR (%) 25.8 (23.2e30.7) 25.4 (22.5e29.2) 0.93 Serial samples of peripheral blood were obtained throughout pregnancy starting Gestational age at 6 0 0.5 at the patient's first visit to our high risk clinic and thereafter every 4 weeks. Blood 9 7 1 samples were collected in tubes containing EDTA, centrifuged at 4 �C for 10 min and delivery (wks) 38.3 (36.7e38.7) 38.8 (36.7e40.2) 0.053 stored at �70 �C until further analysis. Maternal plasma levels of sFlt-1, sEng and Birth weight (grams) PLGF were determined by enzyme-linked immunoassays (R&D Systems, Minneap- 2805 (2525e3345) 3182 (2875e3678) 0.037 olis, MN). All samples were assayed in duplicate at the same time using the same standard curve to minimize interassay variation. The calculated interassay co- Values are expressed as median (interquartile range) or as percentage. efficients of variation for sFlt-1, sEng and PLGF were 3.8%, 6.1% and 7.2% respectively. The calculated intraassay coefficients of variation for sFlt-1, sEng and PLGF were 2.3%, 2.8% and 4.4% respectively. Normality of the data was tested using KolmogoroveSmirnov test. Comparison of continuous variables between the groups was conducted using ManneWhitney U-test or student t-test as appropriate. Chi-square or Fisher exact test were used for comparison of categorical variables. Data are presented as mean ± standard error of the mean (SEM). Significance was accepted at P < 0.05. Statistical analyses were Downloaded for Anonymous User (n/a) at CONSORTIUM MEDICAL LIBRARIES - ISRAEL -Chaim Sheba Medical Center from ClinicalKey.com by Elsevier on May 12, 2018. For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. 165

Y. Yinon et al. / Placenta 36 (2015) 121e124 123 Table 2 Maternal plasma levels of sFlt-1, PLGF, sFlt-1/PLGF ratio and sEng in LMWH-treated and untreated pregnant women. Peptide LMWH group Control group p Value sFlt-1 pg/ml 1309 ± 323 1507 ± 306 0.68 16e27 weeks 2223 ± 359 2687 ± 581 0.5 28e34 weeks 5134 ± 908 4992 ± 1253 0.9 35e40 weeks PLGF pg/ml 342 ± 53 348 ± 47 0.9 16e27 weeks 719 ± 38 558 ± 40 <0.01 28e34 weeks 975 ± 110 511 ± 59 <0.01 35e40 weeks sFlt-1/PLGF ratio 6.5 ± 1.5 5.3 ± 1.4 0.6 16e27 weeks 2.7 ± 0.4 5.8 ± 1.4 <0.05 28e34 weeks 12.9 ± 3.7 <0.05 35e40 weeks 5 ± 1.3 sEng pg/ml 3574 ± 170 0.8 16e27 weeks 3525 ± 157 8113 ± 947 0.75 28e34 weeks 7731 ± 802 10,023 ± 1102 0.7 35e40 weeks 9483 ± 1072 Data are presented as means ± SEM. Fig. 2. The ratio sFlt-1/PLGF throughout gestation in LMWH-treated and untreated pregnant women. Data are presented as means ± SEM.*p < 0.05. levels of sFlt-1. This resulted in a decreased ratio of sFlt-1/PLGF. In contrast to our findings, Rosenberg et al. have showed that heparin is the predominant VEGF inhibitor produced by the placenta in treatment was associated with increased circulating levels of sFlt-1 preeclamptic women [25]. Thus, it is possible that the alleged in the third trimester in 21 pregnant women, who received pro- inconsistency in sFlt-1 levels following heparin treatment reflects phylactic heparin anticoagulation, with no effect on the levels of methodological variance in the determination of different isoforms. PLGF. In vitro, LMWH stimulated sFlt-1 release form placental villous explants, in a dose and time-dependent manner [18]. Despite lack of effect of LMWH on sFlt-1 and sEng circulating Similarly, Drewlo et al. has showed that first trimester placental villi levels, we found that LMWH treatment was associated with exposed to LMWH significantly increased the expression and elevated of PLGF levels starting at the third trimester. In accordance release of sFlt-1 by the syncytiotrophoblast into culture media [17]. with these findings, Drewlo et al. found increased release of PLGF It appears that heparanase, an endogenous enzyme that cleaves from villous explants to the media in response to LMWH, which heparin, facilitates the release of sFlt-1, as its inhibition resulted in was dose-dependent [17]. We did not find a dose-dependent effect reduced amounts of sFlt-1 released from the placental villi [22]. of LMWH on PLGF circulatory levels, most probably since only 5 How can the conflicting findings of our study and previous reports patients in our cohort received treatment dose of 2 mg per kg body regarding the effect of heparin on circulating levels of sFlt-1 be weight while the rest received prophylactic dose. The sFlt-1 to PLGF reconciled? ratio is an index of antiangiogenic activity that reflects changes in the balance between sFlt-1 and PLGF, and has been shown to be While a relatively large body of in-vitro data indicates that more strongly associated with preeclampsia than either measure heparin promotes sFt-1 release from placental villi [17,18,22], in- alone [4]. Moreover, the ratio of sFlt-1/PLGF in the second trimester vivo studies have been limited to very small cohorts [18,22]. can accurately predict severe preeclampsia in high risk women Furthermore, alternative splicing and posttranslational processing [26]. In the current study, LMWH treatment was associated with produce multiple isoforms of sFlt-1 in the circulation of women decreased ratio of sFlt-1/PLGF, which might explain the beneficial with preeclampsia, as well as in uncomplicated pregnancies effect of LMWH in the prevention of several placenta-mediated [23,24]. One of these variants of sFlt-1 is designated sFlt-14, which complications of pregnancy reported in clinical studies [14,20,21]. Fig. 1. Maternal plasma levels of PLGF as a function of gestational age in LMWH-treated and untreated pregnant women. The slopes of best-fit lines of LMWH treatment group (r2 ¼ 0.7) and controls (r2 ¼ 0.28) are significantly different: 44.5 (95% CI 37.9e51.2) vs 17.1 (95% CI 9.8e24.4), p < 0.001. Downloaded for Anonymous User (n/a) at CONSORTIUM MEDICAL LIBRARIES - ISRAEL -Chaim Sheba Medical Center from ClinicalKey.com by Elsevier on May 12, 2018. For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. 166

124 Y. Yinon et al. / Placenta 36 (2015) 121e124 Interestingly, two of our patients treated by LMWH developed [6] Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, et al. Excess placental preeclampsia at 30 and 34 weeks of gestation, and both of them had soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial decreased plasma levels of PLGF and increased sFlt-1/PLGF ratio at dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 28 weeks despite the anticoagulation treatment. 2003;111:649e58. Our findings support the view that heparin exerts its beneficial [7] Venkatesha S, Toporsian M, Lam C, Hanai J, Mammoto T, Kim YM, et al. Soluble effect on placental function not through its anticoagulant action but endoglin contributes to the pathogenesis of preeclampsia. Nat Med 2006;12: possibly by promoting angiogenesis. This is further supported by the 642e9. findings reported by Sobel et al., who showed that exposure of first and second trimester placenta-conditioned media to unfractionated [8] Chaiworapongsa T, Romero R, Espinoza J, Bujold E, Mee Kim Y, Goncalves LF, or LMWH significantly promoted angiogenesis whereas placenta et al. Evidence supporting a role for blockade of the vascular endothelial econditioned media alone from normal first and second trimester growth factor system in the pathophysiology of preeclampsia. Young Inves- explants inhibited angiogenesis [27]. As expected, placenta- tigator Award. Am J Obstet Gynecol 2004;190:1541e7. discussion 1547e50. conditioned media from pregnancies with severe preeclampsia arrested angiogenesis regardless of exposure to LMWH [27]. In [9] McKeeman GC, Ardill JE, Caldwell CM, Hunter AJ, McClure N. Soluble vascular contrast, Rosenberg et al. demonstrated that serum of heparin-treated endothelial growth factor receptor-1 (sFlt-1) is increased throughout gesta- women inhibited both basal and VEGF-induced capillary-like tube tion in patients who have preeclampsia develop. Am J Obstet Gynecol formation, suggesting that heparin actually inhibits angiogenesis [18]. 2004;191:1240e6. Hence, the mechanism underlying the protective effect of heparin against pregnancy complications still needs to be elucidated. [10] Thadhani R, Mutter WP, Wolf M, Levine RJ, Taylor RN, Sukhatme VP, et al. First trimester placental growth factor and soluble fms-like tyrosine kinase 1 and The patients in our study were already on LMWH treatment risk for preeclampsia. J Clin Endocrinol Metab 2004;89:770e5. when enrolled and therefore we could not assess the change in the circulating levels of sFlt-1, PLGF and sEng following initiation of [11] Wang A, Rana S, Karumanchi SA. Preeclampsia: the role of angiogenic factors treatment. Nevertheless, the study groups were well-matched and in its pathogenesis. Physiol e Bethesda 2009;24:147e58. had serial samples collected at similar time points throughout gestation. Therefore, we believe that the significantly elevated cir- [12] Mousa HA, Alfirevic Z. Do placental lesions reflect thrombophilia state in culatory levels of PLGF observed in LMWH treated women women with adverse pregnancy outcome? Hum Reprod 2000;15:1830e3. compared to controls represent a true biological effect of heparin. [13] Kupferminc M, Rimon E, Many A, Maslovitz S, Lessing JB, Gamzu R. Low In summary, we conclude that LMWH treatment during preg- molecular weight heparin versus no treatment in women with previous se- nancy is associated with increased maternal circulatory levels of vere pregnancy complications and placental findings without thrombophilia. PLGF and decreased sFt-1/PLGF ratio. This effect may explain the Blood Coagul Fibrinolysis 2011;22:123e6. protective role heparin might have in preventing placenta- mediated complications. Further investigation is required in order [14] Rey E, Garneau P, David M, Gauthier R, Leduc L, Michon N, et al. Dalteparin for to determine whether the altered balance of sFlt-1 and PLGF during the prevention of recurrence of placental-mediated complications of preg- LMWH treatment is the key factor underlying the beneficial effect nancy in women without thrombophilia: a pilot randomized controlled trial. of heparin treatment during pregnancy. J Thromb Haemost 2009;7:58e64. Disclosure [15] Baczyk D, Dunk C, Huppertz B, Maxwell C, Reister F, Giannoulias D, et al. Bi- potential behaviour of cytotrophoblasts in first trimester chorionic villi. None of the authors have a conflict of interest. Placenta 2006;27:367e74. Acknowledgment [16] Bose P, Black S, Kadyrov M, Bartz C, Shlebak A, Regan L, et al. Adverse effects of lupus anticoagulant positive blood sera on placental viability can be pre- This work was supported by the Ministry of Health (3-7471), vented by heparin in vitro. Am J Obstet Gynecol 2004;191:2125e31. Chief Scientist Office, Israel. [17] Drewlo S, Levytska K, Sobel M, Baczyk D, Lye SJ, Kingdom J. Heparin promotes References soluble VEGF receptor expression in human placental villi to impair endo- thelial VEGF signaling. J Thromb Haemost 2011;9:2486e97. [1] BulletinseObstetrics ACoP. ACOG practice bulletin. Diagnosis and manage- ment of preeclampsia and eclampsia. Number 33, January 2002. Obstet [18] Rosenberg VA, Buhimschi IA, Lockwood CJ, Paidas MJ, Dulay AT, Ramma W, Gynecol 2002;99:159e67. et al. Heparin elevates circulating soluble fms-like tyrosine kinase-1 immu- noreactivity in pregnant women receiving anticoagulation therapy. Circula- [2] Berg CJ, Mackay AP, Qin C, Callaghan WM. Overview of maternal morbidity tion 2011;124:2543e53. during hospitalization for labor and delivery in the United States: 1993e1997 and 2001e2005. Obstet Gynecol 2009;113:1075e81. [19] Brown MA, Lindheimer MD, de Swiet M, Van Assche A, Moutquin JM. The classification and diagnosis of the hypertensive disorders of pregnancy: [3] MacKay AP, Berg CJ, Atrash HK. Pregnancy-related mortality from pre- statement from the International Society for the Study of Hypertension in eclampsia and eclampsia. Obstet Gynecol 2001;97:533e8. Pregnancy (ISSHP). Hypertens Pregnancy 2001;20. IXeXIV. [4] Levine RJ, Lam C, Qian C, Yu KF, Maynard SE, Sachs BP, et al. Soluble endoglin [20] Dodd JM, McLeod A, Windrim RC, Kingdom J. Antithrombotic therapy for and other circulating antiangiogenic factors in preeclampsia. N Engl J Med improving maternal or infant health outcomes in women considered at risk of 2006;355:992e1005. placental dysfunction. Cochrane Database Syst Rev 2013;7:CD006780. [5] Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, et al. Circulating [21] Gris JC, Chauleur C, Faillie JL, Baer G, Mares P, Fabbro-Peray P, et al. Enoxaparin angiogenic factors and the risk of preeclampsia. N Engl J Med 2004;350:672e83. for the secondary prevention of placental vascular complications in women with abruptio placentae. The pilot randomised controlled NOH-AP trial. Thromb Haemost 2010;104:771e9. [22] Sela S, Natanson-Yaron S, Zcharia E, Vlodavsky I, Yagel S, Keshet E. Local retention versus systemic release of soluble VEGF receptor-1 are mediated by heparin-binding and regulated by heparanase. Circ Res 2011;108:1063e70. [23] Rajakumar A, Powers RW, Hubel CA, Shibata E, von Versen-Hoynck F, Plymire D, et al. Novel soluble Flt-1 isoforms in plasma and cultured placental explants from normotensive pregnant and preeclamptic women. Placenta 2009;30:25e34. [24] Thomas CP, Andrews JI, Liu KZ. Intronic polyadenylation signal sequences and alternate splicing generate human soluble Flt1 variants and regulate the abundance of soluble Flt1 in the placenta. FASEB J 2007;21:3885e95. [25] Sela S, Itin A, Natanson-Yaron S, Greenfield C, Goldman-Wohl D, Yagel S, et al. A novel human-specific soluble vascular endothelial growth factor receptor 1: cell-type-specific splicing and implications to vascular endothelial growth factor homeostasis and preeclampsia. Circ Res 2008;102:1566e74. [26] Moore Simas TA, Crawford SL, Solitro MJ, Frost SC, Meyer BA, Maynard SE. Angiogenic factors for the prediction of preeclampsia in high-risk women. Am J Obstet Gynecol 2007;197:244. e241e8. [27] Sobel ML, Kingdom J, Drewlo S. Angiogenic response of placental villi to heparin. Obstet Gynecol 2011;117:1375e83. Downloaded for Anonymous User (n/a) at CONSORTIUM MEDICAL LIBRARIES - ISRAEL -Chaim Sheba Medical Center from ClinicalKey.com by Elsevier on May 12, 2018. For personal use only. No other uses without permission. Copyright ©2018. Elsevier Inc. All rights reserved. 167



‫טרשת נפוצה במהלך התקפי‪ :‬תרופות הדור החדש‬ ‫הרפואה | ‪2014‬‬ ‫מנחה‪ :‬פרופ' ענת אחירון‬ ‫מייסדת פרויקט ח\"ץ‪ ,‬מנהלת המרכז‬ ‫לטרשת נפוצה ואחראית הקתדרה‬ ‫למחלות אוטואימוניות אוניברסיטת ת\"א‬ ‫‪[email protected]‬‬ ‫סער אניס‬ ‫אונ' תל אביב‬ ‫השתתף כסטודנט בפרויקט ח״ץ‬ ‫בין השנים ‪2007-2009‬‬ ‫‪[email protected]‬‬ ‫‪169‬‬

‫הרפואה • כרך ‪ • 153‬חוב' ‪ • 11‬נובמבר ‪2014‬‬ ‫סקירות‬ ‫טרשת נפוצה במהלך התקפי‪ :‬תרופות הדור החדש‬ ‫סער אניס‪1‬‬ ‫במהלך ‪ 20‬השנים האחרונות‪ ,‬הטיפול הקלאסי בטרשת נפוצה במהלך התקפי היה‬ ‫תקציר‪:‬‬ ‫ענת אחירון‪2‬‬ ‫באמצעות הזרקה של שני סוגי תרופות ‪ -‬אינטרפרונים מסוג ביתא וגלטירמר אצטאט‬ ‫(קופקסון)‪ .‬בשנים האחרונות‪ ,‬נכנסו למערך הטיפול תרופות משנות מהלך מחלה‪ ,‬בעלות‬ ‫‪1‬המחלקה לנירולוגיה‪ ,‬מרכז רפואי סוראסקי‪,‬‬ ‫יעילות דומה‪ ,‬ופרופיל בטיחות שאינו נופל ברמתו מהתרופות הקלאסיות‪ .‬התרופות‬ ‫תל אביב‬ ‫החדשות ניתנות בדרך פומית ואף נכללות כקו ראשון לטיפול בחולים‪ .‬כיום‪ ,‬על הקלינאי‬ ‫להתאים בזהירות את התרופה הספציפית לכל חולה על פי פרופיל מחלתו‪ ,‬לאחר שקילת‬ ‫‪2‬המרכז לטרשת נפוצה‪ ,‬מרכז רפואי שיבא‪ ,‬תל‬ ‫הסיכונים והיתרונות של כל תרופה‪ .‬בסקירה זו‪ ,‬נציג את התרופות המאושרות לטיפול‬ ‫השומר‪ ,‬רמת גן‬ ‫בחולי טרשת נפוצה עם מהלך התקפי‪ ,‬לרבות אופן המתן‪ ,‬מנגנון‪ ,‬יעילות‪ ,‬בטיחות‬ ‫והשפעות הלוואי העיקריות של כל טיפול‪ ,‬ונסקור תרופות נוספות הנמצאות בפיתוח‪.‬‬ ‫טרשת נפוצה במהלך התקפי‪.‬‬ ‫מילות מפתח‪:‬‬ ‫‪.Multiple sclerosis; Relapsing remitting multiple sclerosis‬‬ ‫‪:KEY WORDS‬‬ ‫במעקב של חמש שנים התרופה מונעת החמרה במצב הנירולוגי על‬ ‫הקדמה‬ ‫‪658‬‬ ‫פי סולם התקדמות הנכות ‪)EDSS( Expanded Disability Status Scale‬‬ ‫מגוון אפשרויות הטיפול הקיימות למחלת הטרשת הנפוצה גדל‬ ‫ב–‪ 26%‬לעומת אינבו [‪.]2‬‬ ‫במהירות בעשור האחרון‪ .‬התרופה הראשונה המשנה מהלך מחלה‬ ‫‪ :)Biogen, USA( Interferon beta 1a - Avonex‬התרופה אושרה‬ ‫(‪ )Disease modifying treatment‬בעבור חולים עם מהלך התקפי‬ ‫(‪ ,)Relapsing remitting‬אינטרפרון ביתא ‪ 1b‬אושרה לשיווק בשנת‬ ‫על ידי ה–‪ FDA‬לטיפול בחולים עם מהלך התקפי בשנת ‪ .1995‬במחקר®‬ ‫‪ ,1993‬וכיום מצויות תשע תרופות שאושרו על ידי ה–‪Food and( FDA‬‬ ‫אקראי וכפול סמיות שפורסם בשנת ‪ ,]3[ 1996‬הזרקה חד שבועית‬ ‫‪ )drug administration‬לטיפול מכוון בטרשת נפוצה (טבלה ‪.)1‬‬ ‫לתוך השריר של ‪ 30‬מק\"ג במעקב של שנתיים‪ ,‬הובילה לירידה של‬ ‫התרופות המוגדרות כקו ראשון בטיפול הגלטירמר אצטאט‬ ‫‪ 30%‬במספר ההתקפים‪ ,‬לירידה של ‪ 40%‬במספר הנגעים החדשים כפי‬ ‫(קופקסון) ואינטרפרונים מסוג ביתא ניתנות בהזרקה לתת עור או‬ ‫שהודגמו ב–‪ MRI‬ולירידה של ‪ 38%‬בקצב החמרת המחלה על פי סולם‬ ‫לתת שריר בתדירות משתנה של אחת ליום‪ ,‬יום כן‪/‬יום לא‪ ,‬שלוש‬ ‫פעמים בשבוע ואחת לשבוע‪ ,‬בהתאם לתרופה‪ .‬במחקרים רבים בקנה‬ ‫‪ ,EDSS‬זאת בהשוואה לאינבו‪.‬‬ ‫מידה גדול הוכחה יעילות הבטיחות והטיפול בהן‪ .‬עם זאת‪ ,‬אין עוררין‬ ‫‪:Interferon beta -1a - )Merck-Serono, Germany( Rebif‬‬ ‫כי תרופות הניתנות בהזרקה פחות נוחות לחולה‪ .‬התרופות החדשות‬ ‫שאושרו לשיווק בשנים האחרונות מאפשרות טיפול פומי ולתוך‬ ‫התרופה אושרה לטיפול על ידי ה–‪ FDA‬במרץ ‪ 2002‬והוצגה לראשונה®‬ ‫הווריד‪ ,‬עם שיפור ביעילות (‪ )Efficacy‬הטיפול‪ .‬אולם לעיתים התרופות‬ ‫הללו טומנות בחובן השפעות לוואי משמעותיות‪ ,‬ועדיין אין בידינו די‬ ‫במחקר ‪ .]4[ PRISMS‬הוכח כי טיפול במתן זריקה לתת עור במינון ‪22‬‬ ‫ו–‪ 44‬מק\"ג‪ ,‬שלוש פעמים בשבוע למשך שנתיים‪ ,‬מפחית את תדירות‬ ‫נתונים כדי לדעת מהי השפעתן ארוכת הטווח‪.‬‬ ‫ההתקפים הממוצעת השנתית ב–‪ 27%‬ו–‪ ,33%‬בהתאמה‪ ,‬וכן הפחית‬ ‫במאמר זה‪ ,‬נסקור את הטיפולים המקובלים כיום לטרשת נפוצה ‪-‬‬ ‫את מספר הנגעים החדשים ב–‪ MRI‬ב–‪ 3.8%‬לעומת עלייה של ‪10.9%‬‬ ‫הקלאסיים והחדשים‪ .‬המידע נלקח ממחקרי ציר משמעותיים שנערכו‬ ‫לכל תרופה וכולל מנגנון הפעילות המוצע‪ ,‬מידת יעילותן לעומת אינבו‬ ‫במספרם בקבוצת האינבו‪.‬‬ ‫(פלסבו) ולעיתים השוואה של תרופה אחת מול אחרת ‪\" -‬ראש בראש\"‬ ‫במחקר \"ראש בראש\" ייחודי‪ ,‬ה–‪ ,]5[ EVIDENCE‬שפורסם בשנת‬ ‫‪ ,2002‬נערכה השוואה בין טיפול ברביף במינון ‪ 44‬מק\"ג במתן בזריקה‬ ‫וכן השפעות הלוואי הנפוצות‪.‬‬ ‫לתת עור‪ ,‬שלוש פעמים בשבוע‪ ,‬לעומת אבונקס במינון ‪ 30‬מק\"ג בזריקה‬ ‫לשריר אחת לשבוע‪ .‬במחקר הודגמה עדיפות של רביף בהפחתת מספר‬ ‫טיפול באימונומודולטורים בהזרקה‬ ‫ההתקפים ובהפחתה גדולה יותר במספר הנגעים פעילים ב–‪.MRI‬‬ ‫עם זאת‪ ,‬בעקבות הטיפול ברביף הודגמה שכיחות גבוהה יותר של‬ ‫‪ :Interferon beta 1b - )Bayer, Germany( Betaseron‬תרופה משנת®‬ ‫התפתחות נוגדנים כנגד התרופה‪ :‬מבין המטופלים ברביף כ–‪-24%‬‬ ‫‪ 13%‬מהחולים פיתחו נוגדנים מנטרלים כנגד התרופה לעומת ‪5%-2%‬‬ ‫מהלך המחלה שאושרה ביולי ‪ 1993‬על ידי ה–‪ FDA‬לטיפול בטרשת‬ ‫נפוצה במהלך התקפי‪ .‬התרופה היא חלבון הדומה במבנהו לאינטרפרון‬ ‫מהמטופלים באבונקס‪.‬‬ ‫האנושי‪ ,‬אשר מגביר פעילות תאי ‪ T‬מעכבים‪ ,‬מפחית רמות ציטוקינים‬ ‫השפעות הלוואי של אינטרפרונים מסוג ביתא דומות וכוללות‬ ‫פרו–דלקתיים והסננת לימפוציטים למערכת העצבים המרכזית‪ .‬עם‬ ‫כאב ואודם באזור ההזרקה‪ ,‬תסמינים דמויי שפעת‪ ,‬ירידה במספר‬ ‫הליקוציטים ופגיעה בתפקודי הכבד שכללו עלייה ברמות ‪ ALT‬ב–‪37%‬‬ ‫זאת מנגנון הפעולה המדויק אינו ידוע‪.‬‬ ‫מהחולים‪ .‬עלייה זו מתרחשת לרוב בחצי השנה הראשונה של מתן‬ ‫במחקר ציר שפורסם בשנת ‪ ]1[ 1993‬הוכח‪ ,‬כי הזרקת התרופה‬ ‫תכשירי האינטרפרון ולרוב חולפת עצמונית או בעקבות הפחתת מינון‪.‬‬ ‫במתן לתת עור במינון ‪ 250‬מק\"ג לסירוגין יום כן‪/‬יום לא‪ ,‬מפחיתה‬ ‫‪ :Glatiramer acetate - )Teva, Israel( Copaxone‬התרופה‬ ‫את מספר ההתקפים החדים הממוצע השנתי ב–‪ 30%‬לעומת אינבו‪.‬‬ ‫אושרה לשיווק על ידי ה–‪ FDA‬בינואר ‪ .1997‬התרופה היא פולימר של®‬ ‫ארבע חומצות אמיניות המסודרות בצורה אקראית‪ ,‬וניתנת בהזרקה‬ ‫יומית לתת עור במינון של ‪ 20‬מ\"ג‪ .‬פולימר זה דומה במבנהו לחלבון‬ ‫‪170‬‬

‫סקירות‬ ‫הרפואה • כרך ‪ • 153‬חוב' ‪ • 11‬נובמבר ‪2014‬‬ ‫הוכחת טרטוגניות בחיות מעבדה (מומי לב)‪ ,‬יש ליידע נשים בגיל‬ ‫קושר מיאלין (‪ .)Myelin binding protein‬מנגנון המוצע לפעילות‬ ‫התרופה הוא קישור למערכת ה–‪Major Histocompatibility Complex‬‬ ‫הפוריות על הסיכון לעובר (‪.)Class C‬‬ ‫(‪ )MHC‬ותחרות עם אנטיגנים אחרים למיאלין על הקשירה לתאי ‪,T‬‬ ‫‪ :Teriflunomide -)Sanofi, France( Aubagio‬התרופה הפומית‬ ‫ובאופן זה מניעת הרס מיאלין על ידם [‪ .]6‬בנוסף‪ ,‬התרופה משפעלת‬ ‫תאי ‪ T-helper‬מסוג ‪ 2‬הנודדים למוח‪ ,‬ומונעים הרס רקמה במנגנון של‬ ‫השנייה שאושרה בספטמבר ‪ 2012‬על ידי ה–‪ FDA‬לטיפול בחולי טרשת®‬ ‫‪ Bystander suppression‬והפרשת ציטוקינים נוגדי דלקת‪.‬‬ ‫נפוצה עם מהלך התקפי בספטמבר ‪ .2012‬התרופה היא מטבוליט‬ ‫במחקר שפורסם בשנת ‪ 1995‬נמצא‪ ,‬כי התרופה מפחיתה את‬ ‫מספר ההתקפים השנתי ב–‪ 29%‬בהשוואה לאינבו וכן מאטה את קצב‬ ‫פעיל של ‪ Leflunomide‬אשר מעכב ביו–סינתזה של פירימידינים ומונע‬ ‫החמרת המחלה‪ :‬מספר החולים שמצבם החמיר במעל ‪ 1.5‬נקודות‬ ‫בסולם ‪ EDSS‬הייתה כפולה בקבוצת האינבו [‪ .]7‬במחקר נוסף שפורסם‬ ‫התקשרות של תאי ‪ T‬עם תאים מציגי אנטיגן (‪Antigen presenting‬‬ ‫בשנת ‪ 2001‬נמצא‪ ,‬כי התרופה מפחיתה את מספר הנגעים החדשים‬ ‫ב–‪ MRI‬בהשוואה לאינבו [‪ .]8‬השפעות הלוואי של התרופה הן בעיקר‬ ‫‪ .)cells‬במחקר שפורסם בשנת ‪ 2011‬הוכח‪ ,‬כי מתן פומי של התרופה‬ ‫מקומיות באזור ההזרקה עם הופעה של אודם מקומי‪ ,‬ותגובות נוספות‬ ‫כגון כאב בבית החזה‪ ,‬הסמקה‪ ,‬קוצר נשימה‪ ,‬שנובעות מרגישות יתר‬ ‫במינון חד יומי של ‪ 7‬או ‪ 14‬מ\"ג מפחית ב–‪ ,31%‬ללא תלות במינון‪ ,‬את‬ ‫לתרופה והן נדירות‪ .‬נוגדנים התפתחו גם כנגד התרופה גלאטירמר‬ ‫אצטאט‪ ,‬אך ללא משמעות קלינית‪ .‬במחקרי השוואה ראש בראש בין‬ ‫מספר ההתקפים השנתי הממוצע לעומת אינבו [‪ .]13‬במינון הגבוה‪,‬‬ ‫אינטרפרון ביתא לגלטירמר אצטאט‪ ,‬כגון מחקר ‪ BEYOND‬שפורסם‬ ‫בשנת ‪ ]9[ 2009‬או מחקר ‪ REGARD‬בשנת ‪ ,]10[ 2008‬לא נצפתה‬ ‫הפחיתה התרופה בצורה משמעותית סטטיסטית‪ ,‬אך קטנה (‪27%‬‬ ‫עדיפות לאחת התרופות בהשפעתן על ירידה בתדירות ההתקפים‬ ‫מול ‪ ,)21%‬את מספר החולים שמצבם הנירולוגי החמיר במעל נקודה‬ ‫ובעצירת התקדמות המחלה‪.‬‬ ‫בסולם ה–‪ ,EDSS‬וכן הודגמה בבדיקת ‪ MRI‬ירידה של ‪ 18%‬במספר‬ ‫טיפולים אימונומודולטורים פומיים‬ ‫הנגעים החדשים במוח [‪.]14‬‬ ‫‪ :Fingolimode - )Novartis, Switzerland( Gilenya‬התרופה הפומית®‬ ‫השפעות הלוואי הנפוצות של התרופה (מעל ‪ )10%‬הן שלשול‪,‬‬ ‫הראשונה שאושרה בשנת ‪ 2010‬על ידי ה–‪ FDA‬לטיפול בטרשת נפוצה‬ ‫במהלך התקף‪ .‬התרופה היא אנאלוג של ספינגוזין אשר מבצע מודולציה‬ ‫בחילה‪ ,‬הידקקות השיער ועלייה באנזימי כבד ב–‪ .ALT‬התרופה מצויה‬ ‫בקולטן (רצפטור) של ספינגוזין–‪–1‬פוספט‪ ,‬ובאופן זה מונעת יציאת‬ ‫לימפוציטים לדם ההיקפי ומובילה לאגירתם בלשד העצם ובקשריות‬ ‫בקטגוריה של הריון ‪ ,X‬לאחר שהוכח בניסויים בבעלי חיים כי נגרמו‬ ‫הלימפה‪ .‬במחקר ‪ FREEDOMS‬שפורסם בשנת ‪ ]11[ 2010‬הודגם‪ ,‬כי‬ ‫טיפול בפינגולימוד במינון ‪ 1.25‬מ\"ג הוביל לירידה משמעותית של‬ ‫מומים קשים בעובר ולכן אין לקחתה בהריון‪ .‬התרופה מופרשת גם‬ ‫כ–‪ 60%‬בתדירות ההתקפים השנתית לעומת אינבו‪ ,‬ירידה של כ–‪30%‬‬ ‫בסיכוי להחמרת המחלה במשך שנתיים מתחילת הטיפול‪ ,‬וכן ירידה‬ ‫בזרע‪ ,‬ועל כן גבר או אישה המטופלים בתרופה ורוצים להרות‪ ,‬צריכים‬ ‫של ‪ 80%‬בהיווצרות נגעים חדשים במוח שאותרו בבדיקת ‪.MRI‬‬ ‫לעבור סילוק מואץ של התרופה‬ ‫במחקר נוסף שפורסם בשנת ‪ ,2010‬ה–‪ ,TRANSFORMS‬השוו‬ ‫מגופם על ידי נטילת כוליסטראמין ˆ בשנים האחרונות‪ ,‬הצטרפו‬ ‫החוקרים בין מתן פינגולימוד ובין הזרקת ‪ .IFN beta 1a‬בחולים‬ ‫או פחם אקטיבי למשך ‪ 11‬יום‪ ,‬וזאת למערך התרופות כנגד טרשת‬ ‫שטופלו בפינגולימוד הובחנה תדירות התקפים שנתית נמוכה ב–‪51%‬‬ ‫נפוצה במהלך התקפי מספר‬ ‫משום שהתרופה יכולה להישאר‬ ‫לעומת אינטרפרון ביתא וכן ‪ 35%‬פחות נגעים חדשים ב–‪ MRI‬בתקופת‬ ‫תרופות במנגנוני פעולה שונים‪,‬‬ ‫בנסיוב (בסרום) למשך שנתיים‪.‬‬ ‫מעקב של ‪ 12‬חודשים‪ .‬לא נצפו הבדלים משמעותיים סטטיסטית בין‬ ‫התרופות במדד של החמרה במצב המחלה בסולם ‪ EDSS‬במשך שנת‬ ‫אשר היתרון העיקרי של רובן‬ ‫(‪Dimethyl fumarate )BG12‬‬ ‫מעקב [‪ .]12‬החיסרון בתרופה זו הוא טווח השפעות לוואי רחב‪ ,‬כאשר‬ ‫הוא מתן בדרך פומית‪.‬‬ ‫הפחות מסוכנות ויותר נפוצות (מעל ‪ )10%‬כוללות כאבי ראש‪ ,‬תחושה‬ ‫‪ :)Biogen, USA( Tecfidera‬התרופה®‬ ‫דמוית שפעת‪ ,‬שלשול‪ ,‬עלייה באנזימי כבד ושיעול‪ .‬השפעות לוואי‬ ‫נפוצות פחות (<‪ )1%‬כוללות‪ ,‬בצקת מקולרית והפרעות בקצב הלב‬ ‫הפומית השלישית שאושרה מרץ ‪2013‬‬ ‫על ידי ה–‪ FDA‬לטיפול בחולי טרשת ˆ תרופות אלו הניתנות בצורה‬ ‫בעיקר דופק איטי ‪ -‬ברדיאריתמיה‪ ,‬עד ‪.AV block‬‬ ‫נפוצה עם מהלך התקפי‪ .‬תרופה זו פומית הן בעלות פרופיל‬ ‫הופעת ‪ AV block‬היא זמנית ולרוב אי תסמינית‪ ,‬החולפת לאחר ‪24‬‬ ‫היא מולקולה ליפופילית אשר מנגנון יעילות ובטיחות דומה‪,‬‬ ‫שעות‪ ,‬אך לעיתים עלולה לסכן חיים‪ .‬האטה בקצב הלב שכיחה בעיקר‬ ‫פעילותה המשוער הוא הפחתת דלקת שאינו נופל מהתרופות‬ ‫בשעות הראשונות לאחר מתן התרופה בפעם הראשונה‪ .‬במהלך‬ ‫והגנה על תאים נירוניים על ידי שפעול הקלאסיות הניתנות בהזרקה‪.‬‬ ‫הניסויים הקליניים בתרופה היו מספר דיווחים על זיהומים מפושטים‬ ‫של מסלול ‪Nuclear factor related‬‬ ‫של וריצלה זוסטר‪ .‬לכן‪ ,‬לפני תחילת טיפול בפינגולימוד‪ ,‬יש לבצע‬ ‫‪factor 2‬נ(‪ )Nrf2‬והשראה (אינדוקציה) ˆ על הקלינאי להתאים בזהירות‬ ‫בדיקה סרולוגית לווריצלה ואם זו מפורשת כשלילית‪ ,‬לא ניתן לתת‬ ‫את התרופה הספציפית לכל‬ ‫את הטיפול אלא לאחר שהמטופל חוסן‪ .‬בעת מתן התרופה יש צורך‬ ‫של תגובה נוגדת חמצון [‪.]15‬‬ ‫במעקב הכולל‪ )1( :‬ספירת דם ותפקודי כבד; (‪ )2‬אק\"ג (לפני ואחרי‬ ‫בשני מחקרים מקיפים הוכח‪ ,‬חולה על פי פרופיל מחלתו‪,‬‬ ‫מתן התרופה); (‪ )3‬ניטור קצב לב ולחץ דם (כל שעה במשך ‪ 6‬השעות‬ ‫כי התרופה מפחיתה את מספר לאחר שקילת הסיכונים‬ ‫הראשונות לאחר מתן המנה הראשונה); (‪ )4‬בדיקת עיניים להערכת‬ ‫ההתקפים ואת מספר הנגעים והיתרונות של כל תרופה‪.‬‬ ‫בצקת המקולה (לפני תחילת הטיפול ושלושה חודשים אחריו)‪ .‬עקב‬ ‫החדשים במוח כפי שאלה מודגמים‬ ‫בבדיקת ‪ ,MRI‬לעומת טיפול באינבו‪ .‬במחקר ‪ CONFIRM‬שפורסם‬ ‫בשנת ‪ ,2012‬השוו החוקרים בין טיפול ב–‪ BG-12‬במינון של ‪ 480‬מ\"ג‬ ‫או ‪ 720‬מ\"ג ביום‪ ,‬גלטירמר אצטאט ואינבו [‪ .]16‬תוצאות המחקר העלו‪,‬‬ ‫כי התרופה מפחיתה את מספר ההתקפים השנתי ב–‪ 44%‬ו–‪ 51%‬במינון‬ ‫של ‪ 480‬ו–‪ 720‬מ\"ג ביום‪ ,‬בהתאמה‪ ,‬בהשוואה לאינבו‪ ,‬בעוד שבקבוצת‬ ‫החולים שטופלה בגלטירמר אצטאט הובחנה ירידה של ‪ 29%‬בלבד‬ ‫במספר ההתקפים לעומת אינבו‪ .‬עוד נצפתה בבדיקת ‪ MRI‬ירידה‬ ‫משמעותית של ‪ 73%‬במספר הנגעים החדשים ברצף ‪ T2‬לעומת אינבו‪,‬‬ ‫בעוד שבקבוצת החולים שטופלה בגלטירמר אצטאט הובחנה ירידה‬ ‫של ‪ 54%‬בלבד‪.‬‬ ‫במחקר ‪ DEFINE‬שפורסם ב–‪ ,2012‬נערכה השוואה בין מתן ‪BG-12‬‬ ‫במינון ‪ 480‬או ‪ 720‬מ\"ג ביום לעומת אינבו‪ .‬כעבור שנתיים מעקב‬ ‫חלה ירידה משמעותית במספר החולים שחוו התקף (‪ ~26%‬מול‬ ‫‪ 46%‬בהתאמה)‪ .‬השפעות הלוואי הנפוצות (מעל ‪ )10%‬של התרופה‬ ‫הן הסמקה‪ ,‬כאבי בטן‪ ,‬שלשול ובחילה‪ .‬בצורה שכיחה פחות (<‪)10%‬‬ ‫יותר יכולים להופיע לימפופניה‪ ,‬גרד ותיפרחת (‪659 .]17[ )Rash‬‬ ‫‪171‬‬

‫הרפואה • כרך ‪ • 153‬חוב' ‪ • 11‬נובמבר ‪2014‬‬ ‫סקירות‬ ‫טבלה ‪:1‬‬ ‫סיכום מאפייני התרופות המקובלות לטיפול בחולי טרשת נפוצה במהלך התקף‬ ‫השפעות לוואי‬ ‫יעילות בהפחתת‬ ‫יעילות בהאטת‬ ‫יעילות‬ ‫מנגנון‬ ‫אופן מתן‬ ‫תרופה ‪-‬‬ ‫תגובות באזור הזרקה‪ ,‬תסמינים‬ ‫מספר הנגעים‬ ‫התקדמות‬ ‫הפחתת‬ ‫מגביר פעילות תאי‬ ‫לתת עור במינון‬ ‫שם גנרי‬ ‫דמויי שפעת‪ ,‬ירידה בספירת תאי‬ ‫התקפים‬ ‫‪ T‬מעכבים‪ ,‬הורדת‬ ‫‪ 250‬מק\"ג הניתנת‬ ‫ושם מסחרי‬ ‫החדשים ב–‪**MRI‬‬ ‫המחלה בסולם‬ ‫לעומת‬ ‫‪Interferon beta 1a‬‬ ‫הדם הלבנים‪ ,‬פגיעה בתפקוד‬ ‫לעומת אינבו‬ ‫‪ EDSS‬לעומת‬ ‫רמות ציטוקינים‬ ‫יום כן יום לא‬ ‫®‪- Betaseron‬‬ ‫הכבד‪ ,‬פיתוח נוגדנים כנגד התרופה‬ ‫אינבו‬ ‫פרו–דלקתיים‪ ,‬מפחית‬ ‫)בסוגריים מספר‬ ‫אינבו* )בסוגריים‬ ‫‪30%‬‬ ‫תגובות באזור ההזרקה‪ ,‬תגובות‬ ‫שנות מעקב(‬ ‫מספר שנות‬ ‫תנועת לימפוציטים‬ ‫נוספות כגון כאב בבית חזה‪,‬‬ ‫מעקב(‬ ‫למערכת העצבים‬ ‫ירידה של ‪64%‬‬ ‫‪26%‬‬ ‫המרכזית‬ ‫הסמקה‪ ,‬קוצר נשימה‪ ,‬דפיקות לב‬ ‫בנפח הנגעים ב–‬ ‫(פלפיטציות) וחרדה‬ ‫‪ MRI‬לעומת אינבו‬ ‫‪38% 30%‬‬ ‫לתוך השריר‬ ‫‪Interferon beta 1a‬‬ ‫במעקב של ‪ 4‬שנים‬ ‫במינון ‪ 30‬מק\"ג‬ ‫®‪-Avonex‬‬ ‫כאבי ראש‪ ,‬תחושה דמוית שפעת‪,‬‬ ‫‪ 27%‬ו–‪- 33%‬‬ ‫‪Interferon‬‬ ‫שלשול‪ ,‬עלייה באנזימי כבד זיהומי‬ ‫ירידה של ‪40%‬‬ ‫במינון‬ ‫הניתנת חד‬ ‫‪beta-1a‬‬ ‫בנפח הנגעים ב–‬ ‫שבועית‬ ‫®‪Rebif‬‬ ‫שלבקת‪ ,‬סרטן עור‪/‬שד‪ ,‬הפרעות‬ ‫‪ MRI‬לעומת אינבו‬ ‫נמוך וגבוה‬ ‫בקצב הלב‬ ‫במעקב של שנתיים‬ ‫בהתאמה‬ ‫לתת עור במינון‬ ‫‪ 22‬ו–‪ 44‬מק\"ג ‪3‬‬ ‫תסמיני מערכת עיכול‪ ,‬הידקקות‬ ‫ירידה של ‪1.2%‬‬ ‫פעמים בשבוע‬ ‫השיער‪ ,‬פגיעה בתפקודי כבד‪.‬‬ ‫ו–‪ 3.8%‬בהתאמה‬ ‫קטגורית הריון ‪ ,X‬צורך בסילוק‬ ‫בנפח הנגעים ב–‪MRI‬‬ ‫‪***46%‬‬ ‫לתת עור במינון מונע הרס של המיאלין ‪29%‬‬ ‫‪Glatiramer‬‬ ‫‪30%‬‬ ‫‪ 20‬מ\"ג הניתנת חד ע\"י תחרות עם‬ ‫‪acetate‬‬ ‫התרופה מהגוף לפני ניסיון כניסה‬ ‫לעומת עליה ב‬ ‫אנטיגנים של מיאלין‬ ‫יומית‬ ‫להריון (גם לגברים עקב המצאות‬ ‫‪ 10.9%‬באינבו‬ ‫על הקשירה לתאי ‪T‬‬ ‫®‪Copaxone‬‬ ‫‪11%‬‬ ‫®‪Fingolimode‬‬ ‫התרופה בזרע)‬ ‫‪80%‬‬ ‫מניעת נדידה של ‪60%‬‬ ‫פומי במינון ‪1.25‬‬ ‫הסמקה‪ ,‬תסמיני מערכת עיכול‪,‬‬ ‫‪18%‬‬ ‫לימפוציטים מקשריות‬ ‫מ\"ג‬ ‫לימפה‬ ‫לימפופניה‪ ,‬גרד ותיפרחת‬ ‫‪73%‬‬ ‫כאבי ראש‪ ,‬שלשול‪ ,‬עלייה באנזימי‬ ‫‪30%‬‬ ‫פומי במינון של ‪ 7‬מעכב ביו–סינתזה של ‪( 31%‬ללא ‪26%‬‬ ‫‪Teriflunomide‬‬ ‫תלות‬ ‫או ‪ 14‬מ\"ג הניתנת פירימידינים ומונע‬ ‫®‪Aubagio‬‬ ‫כבד‪ ,‬תסמינים דמויי שפעת‪ ,‬יתר‬ ‫‪83%‬‬ ‫במינון)‬ ‫התקשרות של תאי ‪T‬‬ ‫חד יומית‬ ‫לחץ דם‪ ,‬ברדיקרדיה‪ ,‬דיכאון‪,‬‬ ‫עם תאים מציגי אנטיגן‬ ‫‪( 18%‬לעומת קבוצת‬ ‫ירידה בספירת תאי הדם הלבנים‬ ‫‪)Interferon 1-a‬‬ ‫‪ 44%‬ו– ‪24%‬‬ ‫שפעול גלותטיון‬ ‫פומי במינון‬ ‫‪Dimethyl‬‬ ‫‪PML )progressive multifocal‬‬ ‫‪ 51%‬במינון‬ ‫והפעלת חלבונים נוגדי‬ ‫‪ 240‬מ\"ג הניתנת‬ ‫)‪fumarate (BG12‬‬ ‫נמוך וגבוה‬ ‫פעמיים או שלוש‬ ‫(‪ ,leukoencephalopathy‬תגובה‬ ‫בהתאמה‬ ‫דלקת‪ ,‬השפעה מגנה‬ ‫®‪Tecfidera‬‬ ‫אלרגית‪ ,‬חרדה‪ ,‬פרינגיטיס‪ ,‬בצקת‬ ‫על תאים נירונליים‬ ‫פעמים ביום‬ ‫®‪****Laquinimod‬‬ ‫‪30% 23%‬‬ ‫הפעלת קולטן‬ ‫פומי במינון ‪0.6‬‬ ‫היקפית‬ ‫‪sphingosine‬‬ ‫מ\"ג הניתנת חד‬ ‫‪Natalizumab‬‬ ‫‪26% 68%‬‬ ‫®‪Tysabry‬‬ ‫כאב ראש‪ ,‬חום‪ ,‬בחילה‪ ,‬תיפרחת‪,‬‬ ‫(‪ 1-phosphate )S1P‬דבר‬ ‫יומית‬ ‫זיהומי שלבקת (הרפס)‪ ,‬תגובות‬ ‫‪( 30%‬לעומת‬ ‫‪54%‬‬ ‫המוביל לירידה בנדידה‬ ‫‪Alemtuzumab‬‬ ‫אוטואימוניות‬ ‫קבוצת ‪Interferon‬‬ ‫(לעומת‬ ‫עירוי לתוך הווריד‬ ‫®‪*****Lemtrada‬‬ ‫קבוצת‬ ‫של לימפוציטים‬ ‫במינון של ‪300‬‬ ‫‪)1-a‬‬ ‫‪Interferon‬‬ ‫מקשריות לימפה‬ ‫מ\"ג הניתנת אחת‬ ‫‪)1-a‬‬ ‫נוגדן חד שבטי‬ ‫ל‪ 4-‬שבועות‬ ‫(מונוקלונלי) רקומביננטי‬ ‫‪ 12‬מ\"ג במתן לתוך‬ ‫המונחה כנגד ‪alpha-4‬‬ ‫הווריד ל–‪ 5‬ימים‬ ‫‪ integrins‬ועל ידי כך‬ ‫ובהמשך מתן לתוך‬ ‫מונע כניסת תאי דלקת‬ ‫הווריד חד שנתי‬ ‫למערכת העצבים‬ ‫למשך ‪ 3‬ימים‬ ‫המרכזית‬ ‫נוגדן חד שבטי‬ ‫הומני הנקשר לחלבון‬ ‫‪ CD52‬ומוריד רמות‬ ‫לימפוציטים ‪T,B‬‬ ‫* התקדמות המחלה (‪ )Disability progression‬מוגדרת על ידי החמרה של ‪ 1.0‬נקודה ומעלה בסולם ה–‪ EDSS‬במעקב של שנתיים‬ ‫** ברצף ‪.T2‬‬ ‫*** התקדמות המחלה במקרה זה הוגדרה על ידי התדרדרות של ‪ 1.5‬נקודות בסולם ה–‪ EDSS‬במעקב של ‪ 140‬שבועות (~‪ 2.5‬שנים)‬ ‫**** אינה מאושרת לטיפול‪.‬‬ ‫**‪ 6**6*0‬אינה מאושרת על ידי ה–‪ ,FDA‬אלא על ידי הסוכנות האירופית לתרופות בלבד‪.‬‬ ‫‪172‬‬

‫הרפואה • כרך ‪ • 153‬חוב' ‪ • 11‬נובמבר ‪2014‬‬ ‫סקירות‬ ‫‪ .1,000‬השפעות לוואי נוספות כוללות עייפות‪ ,‬תגובה אלרגית וחרדה‪.‬‬ ‫‪ :)Teva, Israel( Laquinimod‬תרופה פומית שעברה ניסוי קליני®‬ ‫‪6621‬‬ ‫‪ :)Sanofi, France( Alemtuzumab-Lemtrada‬התרופה אינה‬ ‫אולם טרם אושרה לטיפול‪ .‬מנגנון התרופה המוצע הוא הפחתה‬ ‫מאושרת על ידי ה–‪ FDA‬לטיפול בחולי טרשת נפוצה‪ ,‬ונדחתה בדצמבר®‬ ‫בהסננת תאי דלקת למערכת העצבים המרכזית‪ ,‬הפחתת דה–מיאלינציה‬ ‫ומניעה של פגיעה אקסונאלית על ידי הפחתת רמות ציטוקינים שונים‪,‬‬ ‫‪ 2013‬בדרישה לביצוע מחקרים קליניים נוספים להוכחת יעילותה‪ .‬עם‬ ‫כגון אינטרליקין ‪ .]18[ 17‬הוכח‪ ,‬כי המולקולה מפחיתה הפרשת‬ ‫זאת‪ ,‬היא אושרה לטיפול על ידי הסוכנות האירופית לתרופות בשנת‬ ‫ציטוקינים פרו–דלקתיים‪ ,‬מגבירה הפרשת ציטוקינים נוגדי דלקת‬ ‫‪ .2013‬המולקולה היא נוגדן חד שבטי אנושי שגורם לירידה בתאי ‪NK ,T‬‬ ‫בתאי דם לבנים ומעלה רמות ‪Brain-Derived Neurotrophic Factor‬‬ ‫ומונוציטים המבטאים ‪ CD52‬על ידי קשירה ישירה לחלבון זה ‪ -‬המובילה‬ ‫(‪ )BDNF‬בנסיוב‪ ,‬שידוע כי הוא תורם להישרדות נירונים ומסייע‬ ‫להרס התא‪ .‬התרופה ניתנת בעירוי במשך חמישה ימים רצופים‪ ,‬ובהמשך‬ ‫לגדילה והתמיינות של נירונים חדשים בסינפסות [‪.]19‬‬ ‫במתן חד שנתי של עירוי לתוך הווריד במשך שלושה ימים רצופים‪.‬‬ ‫יעילות התרופה הוערכה בשני מחקרי ציר‪ :‬בשנת ‪ 2012‬פורסם‬ ‫מחקרי הציר בתרופה זו כוללים ‪ ,]26[ CARE-MS I‬שבו הושווה‬ ‫מחקר ‪ ]20[ ALLEGRO‬שהוכח בו‪ ,‬כי מתן התרופה במינון פומי ‪0.6‬‬ ‫מתן הנוגדן לאינטרפרון ביתא ‪ .1a‬שיעור של ‪ 22%‬מהחולים שטופלו‬ ‫מ\"ג הפחית את מספר ההתקפים הממוצע השנתי ב–‪ 23%‬לעומת אינבו‪,‬‬ ‫באלמטוזומאב חוו התקף במהלך שנתיים של טיפול לעומת ‪40%‬‬ ‫הפחית ב–‪ 30%‬את הסיכון להחמרת המחלה בסולם ‪ EDSS‬וב–‪ 30%‬את‬ ‫מהחולים בקבוצת האינטרפרון‪ .‬כמו כן‪ ,‬התרופה הפחיתה (לעומת‬ ‫מספר הנגעים החדשים במוח בבדיקת ב–‪ .T2 MRI‬במחקר שני שפורסם‬ ‫קבוצת האינטרפרון) את תדירות ההתקפים השנתית הממוצעת‬ ‫בשם ‪ ]21[ BRAVO‬נערכה השוואה בין לקווינימוד ואינטרפרון ביתא ‪1a‬‬ ‫ב–‪ ,54%‬את הסיכון להחמרת המחלה ב–‪ 30%‬ואת מספר הנגעים‬ ‫לאינבו (התרופות לא הושוו בינן לבין עצמן)‪.‬‬ ‫החדשים במוח ברצף ‪ T2‬ב–‪ MRI‬ב–‪.18%‬‬ ‫בניתוח ראשוני התרופה לא הובילה לירידה משמעותית סטטיסטית‬ ‫במחקר ‪ ]27[ CARE-MS II‬הוכחה יעילותה של התרופה בהפחתת‬ ‫במספר ההתקפי השנתי הממוצע בהשוואה לאינבו‪ ,‬בעוד שאינטרפרון‬ ‫מספר ההתקפים ובמניעת החמרה במצבם של חולים שהטיפול‬ ‫הצליח להגיע למשמעות סטטיסטית‪ .‬בניתוח עם נרמול נוסף של‬ ‫הראשוני בהם בתרופות אינטרפרון ביתא ‪ 1a‬או גלטירמר אצטאט‪,‬‬ ‫הנתונים‪ ,‬הושגה משמעות סטטיסטית בהפחתת מספר ההתקפים‬ ‫כשל‪ .‬השפעות הלוואי העיקריות הן תגובות בזמן מתן העירוי‬ ‫השנתי בעקבות הטיפול בתרופה ‪ -‬ירידה של ‪ 22%‬בהשוואה לאינבו‪.‬‬ ‫(‪ 90%‬מהחולים) שכללו כאב ראש‪ ,‬חום‪ ,‬בחילה ותיפרחת‪ ,‬ולכן מתן‬ ‫השפעות הלוואי הנפוצות כוללות כאבי ראש (‪ ,)10%‬שלשול (‪,)12%‬‬ ‫התרופה משולב עם מתן סטרואידים ותרופות נוגדות אלרגיה‪ .‬כמו‬ ‫כן דווח על שיעור גבוה של זיהומים בעיקר בשלבקת (הרפס) ב–‪18%‬‬ ‫עלייה באנזימי כבד (‪ ,)14%‬כאבי גב ותסמינים דמויי שפעת (‪.)12%‬‬ ‫מהחולים שהוביל לטיפול מונע באציקלוביר בזמן מתן העירוי ו–‪28‬‬ ‫ימים לאחריו; שיעור גבוה של תגובות אוטואימוניות המערבות את‬ ‫נוגדנים חד שבטיים (מונוקלונאליים)‬ ‫בלוטת התריס ב–‪ 18%-16%‬מהחולים; והיארעות תרומבוציטופניה‬ ‫‪ :)Biogen, USA( Natalizumab - Tysabri‬התרופה אושרה על ידי®‬ ‫אוטואימונית העלולה להיות קטלנית ב–‪ 1%‬מהחולים [‪.]28‬‬ ‫‪ :Polymerase 1 inhibitor‬התרופה נמצאת בפיתוח על ידי המרכז‬ ‫ה–‪ FDA‬לטיפול בחולי טרשת נפוצה בשנת ‪ 2004‬ולאחר מכן הורדה‬ ‫לטרשת נפוצה‪ ,‬במרכז הרפואי שיבא‪ ,‬תל השומר‪ .‬פיתוח התרופה‬ ‫מהמדפים‪ ,‬כתוצאה משלושה אירועי מוות של חולים מנגיף ‪,JC‬‬ ‫מבוסס על מחקר בפלטפורמת שבבי ביטוי גנים‪ ,‬שבו הושוו חולי טרשת‬ ‫שהביא להתפתחות ‪Progressive Multifocal Leukoencephalopathy‬‬ ‫נפוצה במהלך טב (‪ )Benign‬לחולים במהלך התקפי ופורסם בשנת ‪2012‬‬ ‫(‪ .)PML‬התרופה אושרה לטיפול פעם נוספת בשנת ‪ ,2006‬לאחר‬ ‫[‪ .]29‬תוצאות ההשוואה הצביעו כי מסלול ‪ Polymerase 1‬מדוכא בחולים‬ ‫בחינה מחודשת ונתינת התרופה תחת מעקב לגורמי הסיכון ל–‪.PML‬‬ ‫במהלך טב בהשוואה למהלך התקפי‪ ,‬והוצע כי דיכוי המסלול יכול‬ ‫המולקולה היא נוגדן חד שבטי רקומביננטי המונחה כנגד ‪Alpha-4‬‬ ‫לשנות את מהלך מחלה‪ .‬במחקר עוקב שנערך בעכברים בפלטפורמה‬ ‫‪ .integrins‬האינטגרינים הללו מצויים על פני שטח הלימפוציט‬ ‫של ‪ .]30[ )EAE( Experimental Autoimmune Encephalomyelitis‬הודגם‪,‬‬ ‫והמונוציט‪ ,‬וממלאים תפקיד חשוב בהיצמדותם לאנדותל הווסקולרי‬ ‫כי התרופה המעכבת מסלול זה מנעה לחלוטין את התפתחות המחלה‬ ‫בעכברים‪ ,‬ועיכבה את התפתחות המחלה בעכברים שהראו סימני‬ ‫ולמעברם דרך המחסום דם–מוח למוח [‪.]22‬‬ ‫התרופה ניתנת בעירוי לתוך הווריד במינון של ‪ 300‬מ\"ג אחת‬ ‫קליניים של המחלה‪.‬‬ ‫לארבעה שבועות במשך שעה‪ .‬שני מחקרי של נטליזומאב פורסמו‬ ‫בשנת ‪ .2006‬במחקר ‪ ,]23[ AFFIRM‬הוכח כי התרופה מפחיתה את‬ ‫דיון וסיכום‬ ‫מספר ההתקפים הממוצע השנתי ב–‪ 68%‬בהשוואה לאינבו‪ .‬במחקר‬ ‫‪ ]24[ SENTINAL‬נלקחו חולים שטופלו באינטרפרון ביתא ‪ a1‬שלא הגיבו‬ ‫מסקירה זו עולה‪ ,‬כי בשנים האחרונות גדל סל התרופות לטיפול בחולי‬ ‫לטיפול (התבטא בהמשך התקפים ובהחמרת המחלה) וסווגו לשתי‬ ‫טרשת נפוצה במהלך התקפי‪ .‬בעקבות התרופות החדשות חל שיפור‬ ‫קבוצות‪ )1( :‬אינבו (‪ )2‬נטליזומאב‪ ,‬במקביל להמשך טיפול באינטרפרון‬ ‫ביעילות הטיפול‪ ,‬ללא התפשרות בבטיחות התרופה‪ ,‬וחל מעבר הדרגתי‬ ‫ביתא ‪ .a1‬המחקר נעצר כחודש טרם סיומו‪ ,‬עקב היארעות של ‪PML‬‬ ‫מהתרופות הקלאסיות שניתנו דרך הווריד או לתת עור לתרופות‬ ‫בשני חולים בזרוע הטיפולית בנטליזומאב‪ .‬הטיפול הביא לירידה של‬ ‫‪ 83%‬במספר הנגעים החדשים החדשים‪/‬הגדלים במוח ברצף ‪ T2‬ב–‪.MRI‬‬ ‫הניתנות בדרך פומית‪.‬‬ ‫תוצאות שני המחקרים הצביעו על כך שהתרופה היא היעילה ביותר‬ ‫עם זאת‪ ,‬לנוכח ההיצע הרחב של תרופות העומד לרשותו של הקלינאי‪,‬‬ ‫כיום בדיכוי הפעילות הדלקתית בטרשת נפוצה‪ .‬הסיכון בתרופה זו‬ ‫ללא תרופה אחת המראה עליונות‪ ,‬עומדת בפניו החלטה לא פשוטה‬ ‫נעוץ בסיכון לחלות ב–‪ ,PML‬כאשר גורמי הסיכון לפתח את המחלה‬ ‫בבחירת התרופה המתאימה לחולה‪ .‬מכאן כי על הנירולוג להתאים‬ ‫תחת הטיפול בטיסברי הם‪ )1( :‬נוכחות נוגדנים לנגיף ‪)2( ;JC virus‬‬ ‫טיפול מדכא חיסון (אימונוסופרסיבי) קודם; (‪ )3‬משך נטילת הטיפול‪.‬‬ ‫בזהירות את התרופה הספציפית לכל חולה‪ ,‬על פי פרופיל מחלתו‪ ,‬‬ ‫בחולים שהם סרו–נגטיביים לנגיף‪ ,‬הסיכון לפתח ‪ PML‬בנטילת התרופה‬ ‫לאחר שקילת הסיכונים והיתרונות של כל תרופה‪.‬‬ ‫קטן מ–‪ 0.09‬לכל ‪ 1,000‬מטופלים [‪ .]25‬לחולים סרו–פוזיטיביים לנגיף‪,‬‬ ‫הסיכון לפתח ‪ PML‬תוך שנתיים מתחילת הטיפול הוא ‪ 0.56‬לכל ‪1,000‬‬ ‫מחבר מכותב‪ :‬סער אניס‬ ‫מטופלים‪ ,‬ואם הם מטופלים במשך ‪ 4-2‬שנים‪ ,‬עולה הסיכון ל‪ 4.6-‬לכל‬ ‫רחוב מונטיפיורי ‪ ,1‬קריית אונו‬ ‫דוא\"ל‪[email protected] :‬‬ ‫‪173‬‬

2014 ‫ • נובמבר‬11 '‫ • חוב‬153 ‫הרפואה • כרך‬ ‫סקירות‬ ‫ביבליוגרפיה‬ 1. Interferon beta-1b is sclerosis: results of a phase 14. He D, Xu Z, Dong S & al, sclerosis. Ann Neurol, effective in relapsing- III multicenter, double- Teriflunomide for multiple 2006;59:748-54. remitting multiple blind placebo-controlled sclerosis. Cochrane sclerosis. I. Clinical trial. The Copolymer 1 Database Syst Rev, 23. Polman CH, O’Connor results of a multicenter, Multiple Sclerosis Study 2012;12:CD009882. PW, Havrdova E & al, A randomized, double- Group. Neurology, randomized, placebo- blind, placebo-controlled 1995;45:1268-76. 15. Scannevin RH, Chollate S, controlled trial of trial. The IFNB Multiple Jung MY & al, Fumarates natalizumab for relapsing Sclerosis Study Group. 8. Comi G, Filippi M & Wolinsky promote cytoprotection multiple sclerosis. N Engl J Neurology, 1993;43:655-61. JS, European/Canadian of central nervous system Med, 2006;354:899-910. multicenter, double-blind, cells against oxidative 2. Interferon beta-1b in the randomized, placebo- stress via the nuclear factor 24. Rudick RA, Stuart treatment of multiple controlled study of the )erythroid-derived 2)-like 2 WH, Calabresi PA & sclerosis: final outcome of effects of glatiramer acetate pathway. J Pharmacol Exp al, Natalizumab plus the randomized controlled on magnetic resonance Ther, 2012;341:274-84. interferon beta-1a for trial. The IFNB Multiple imaging--measured disease relapsing multiple Sclerosis Study Group activity and burden in 16. Fox RJ, Miller DH, Phillips sclerosis. N Engl J Med, and The University of patients with relapsing JT & al, Placebo-controlled 2006 ;354:911-23. British Columbia MS/MRI multiple sclerosis. European/ phase 3 study of oral BG-12 Analysis Group. Neurology, Canadian Glatiramer or glatiramer in multiple 25. Bloomgren G, Richman S, 1995;45:1277-85. Acetate Study Group. Ann sclerosis. N Engl J Med, Hotermans C & al, Risk of Neurol, 2001 ;49:290-7. 2012;367:1087-97. natalizumab-associated 3. Jacobs LD, Cookfair progressive multifocal DL, Rudick RA & al, 9. O’Connor P, Filippi M, 17. Gold R, Kappos L, leukoencephalopathy. N Engl Intramuscular interferon Arnason B & al, 250 Arnold DL & al, Placebo- J Med, 2012;366:1870-80. beta-1a for disease microg or 500 microg controlled phase 3 study progression in relapsing interferon beta-1b versus of oral BG-12 for relapsing 26. Cohen JA, Coles AJ, Arnold multiple sclerosis. 20 mg glatiramer acetate multiple sclerosis. N Engl J DL & al, Alemtuzumab The Multiple Sclerosis in relapsing-remitting Med, 2012;367:1098-107. versus interferon beta 1a Collaborative Research multiple sclerosis: a as first-line treatment for Group )MSCRG). Ann prospective, randomised, 18. Wegner C, Stadelmann patients with relapsing- Neurol, 1996;39:285-94. multicentre study. Lancet C, Pfortner R & al, remitting multiple Neurol, 2009;8:889-97. Laquinimod interferes sclerosis: a randomised 4. Randomised double-blind with migratory capacity controlled phase 3 trial. placebo-controlled study 10. Mikol DD, Barkhof F, Chang of T cells and reduces Lancet, 2012;380:1819-28. of interferon beta-1a P & al, Comparison of IL-17 levels, inflammatory in relapsing/remitting subcutaneous interferon demyelination and acute 27. Coles AJ, Twyman multiple sclerosis. beta-1a with glatiramer axonal damage in mice with CL, Arnold DL & al, PRISMS )Prevention of acetate in patients with experimental autoimmune Alemtuzumab for patients Relapses and Disability relapsing multiple sclerosis encephalomyelitis. with relapsing multiple by Interferon beta-1a )the REbif vs Glatiramer J Neuroimmunol, sclerosis after disease- Subcutaneously in Multiple Acetate in Relapsing MS 2010;227:133-43. modifying therapy: a Sclerosis) Study Group. Disease [REGARD] study): randomised controlled Lancet, 1998;352:1498-504. a multicentre, randomised, 19. Bruck W & Wegner C, phase 3 trial. Lancet, 2012 parallel, open-label trial. Insight into the mechanism 24;380:1829-39. 5. Panitch H, Goodin DS, Lancet Neurol, 20087:903-14. of laquinimod action. J Francis G & al, Randomized, Neurol Sci, 2011;306:173-9. 28. Willis M & Robertson NP, comparative study of 11. Kappos L, Radue EW, Drug safety evaluation of interferon beta-1a treatment O’Connor P & al, A 20. Comi G, Jeffery D, Kappos alemtuzumab for multiple regimens in MS: The placebo-controlled L & al, Placebo-controlled sclerosis. Expert Opin EVIDENCE Trial. Neurology, trial of oral fingolimod trial of oral laquinimod for Drug Saf, 2014;13:1115-24. 2002;59:1496-506. in relapsing multiple multiple sclerosis. N Engl J sclerosis. N Engl J Med, Med, 2012;366:1000-9. 29. Achiron A, Feldman 6. Arnon R & Aharoni R, 2010;362:387-401. A, Magalashvili D & Mechanism of action 21. BRAVO study: Laquinimod al, Suppressed RNA- of glatiramer acetate 12. Cohen JA, Barkhof F, Comi double blind placebo polymerase 1 pathway is in multiple sclerosis G & al, Oral fingolimod or controlled study in RRMS associated with benign and its potential for the intramuscular interferon patients with a rater blinded multiple sclerosis. PLoS development of new for relapsing multiple reference arm of interferon One, 2012;7:e46871. applications. Proc Natl sclerosis. N Engl J Med, β-1a )Avonex®). http:// Acad Sci U S A, 2004;101 2010;362:402-15. clinicaltrials.gov/ct2/ 30. Achiron A, Mashiach )Suppl 2):14593-8. show/study/NCT00605215 R, Zilkha-Falb R & al, 13. O’Connor P, Wolinsky )Accessed on March 31, 2012. Polymerase I pathway 7. Johnson KP, Brooks BR, JS, Confavreux C & al, inhibitor ameliorates Cohen JA & al, Copolymer Randomized trial of oral 22. Niino M, Bodner C, Simard experimental autoimmune 1 reduces relapse rate and teriflunomide for relapsing ML & al, Natalizumab encephalomyelitis. J improves disability in multiple sclerosis. N Engl J effects on immune cell Neuroimmunol,2013;2:91-7. relapsing-remitting multiple Med, 2011;365:1293-303. responses in multiple 6642 174

Phosphorylcholine-tuftsin compound prevents development of dextransulfate-sodium-salt induced murine colitis: implications for the treatment of human inflammatory bowel disease Journal of Autoimmunity | 2015 ‫ פרופ' מרים בלנק‬:‫מנחה‬ ‫מרכז למחלות אוטואימוניות‬ [email protected] ‫ירדן לחניש‬ ‫אונ' תל אביב‬ ‫השתתף כסטודנט בפרויקט ח״ץ‬ 2012-2013 ‫בין השנים‬ [email protected] 175

Journal of Autoimmunity 56 (2015) 111e117 Contents lists available at ScienceDirect Journal of Autoimmunity journal homepage: www.elsevier.com/locate/jautimm Phosphorylcholine-tuftsin compound prevents development of dextransulfate-sodium-salt induced murine colitis: Implications for the treatment of human inflammatory bowel disease Dana Ben-Ami Shor a, b, 1, Tomer Bashi b, 2, Jordan Lachnish b, Mati Fridkin c, Giorgia Bizzaro b, Iris Barshak d, Miri Blank b, Yehuda Shoenfeld b, e, * a Department of Gastroenterology, Sheba Medical Center affiliated to Sackler Faculty of Medicine, Tel-Aviv University, Israel b Zabludowicz Center for Autoimmune Diseases, Sheba Medical Center affiliated to Sackler Faculty of Medicine, Tel-Aviv University, Israel c Department of Organic Chemistry, The Weizmann Institute of Sciences, Rehovot, Israel d Institute of Pathology, Sheba Medical Center, affiliated to Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel e Incumbent of the Laura Schwarz-Kipp Chair for Research of Autoimmune Diseases, Tel-Aviv University, Israel article info abstract Article history: Improved clinical findings of inflammatory bowel disease (IBD) upon treatment with helminthes and Received 13 October 2014 their ova were proven in animal models of IBD and in human clinical studies. The immunomodulatory Received in revised form properties of several helminthes were attributed to the phosphorylcholine (PC) molecule. We assessed 12 November 2014 the therapeutic potential of tuftsin-PC conjugate (TPC) to attenuate murine colitis. Colitis was induced by Accepted 12 November 2014 Available online 03 December 2014 Dextransulfate-Sodium-Salt (DSS) in drinking water. TPC was given by daily oral ingestion (50 mg/0.1 ml/ Keywords: mouse or PBS) starting at day �2. Disease activity index (DAI) score was followed daily and histology of Colitis the colon was performed by H&E staining. Analysis of the cytokines profile in distal colon lysates was Helminthes performed by immunoblot. Treatment of DSS induced colitis with TPC prevented the severity of colitis, Inflammatory bowel disease including a reduction in the DAI score, less shortening of the colon and less inflammatory activity in Phosphorylcholine histology. The immunoblot showed that the colitis preventive activity of TPC was associated with Tuftsin Worms downregulation of colon pro-inflammatory IL-1b, TNFa and IL-17 cytokines expression, and enhance- ment of anti-inflammatory IL-10 cytokine expression. In the current study, we demonstrated that TPC treatment can prevent significantly experimental colitis induction in naïve mice. We propose the TPC as a novel potential small synthetic molecule to treat colitis. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction methotrexate, calcineurin inhibitors and anti-TNF often causes se- vere side effects [1]. Inflammatory bowel disease (IBD) is comprised of two major phenotypes, Crohn's disease (CD) and ulcerative colitis (UC). The In Western countries, a significant increase in the prevalence of progress in deciphering the molecular mechanisms underlying the autoimmune and auto-inflammatory syndromes is strongly corre- pathogenesis of IBD and other autoimmune diseases, has led to the lated with improved sanitation and hygiene. Unsurprisingly, a development of new-targeted biological treatments [1]. Never- strong correlation was reported between high prevalence of para- theless, existing therapies for IBD such as thiopurines, sitic worms (helminthes) in certain geographic areas and immune protection from atopic, autoimmune, and auto inflammatory dis- * Corresponding author. Zabludowicz Center for Autoimmune Diseases, Sheba eases [2e5]. Through co-evolution of the helminthes with their Medical Center affiliated to Sackler Faculty of Medicine, Tel-Aviv University, Tel- hosts, the helminthes were able to modulate inflammatory Hashomer, 52621, Israel. Tel.: þ972 3 5308070, þ972 526669020 (moblie); response in the host in order to promote parasite survival. This may fax: þ972 3 5352855. have also generated a predisposition in the host towards the development of autoimmunity in the absence of infection [6]. E-mail address: [email protected] (Y. Shoenfeld). 1 This work was performed in fulfillment of the requirements for the internal These observations led to the successful application of hel- medicine thesis, Sackler Faculty of Medicine, Tel Aviv University, Israel. minthes and their ova, which resulted in ameliorated clinical and 2 This work was performed in partial fulfillment of the requirements for PhD laboratory findings in a large number of autoimmune diseases, thesis, Sackler Faculty of Medicine, Tel Aviv University, Israel. including multiple sclerosis [7,8], torheumatoid arthritis [9,10], type http://dx.doi.org/10.1016/j.jaut.2014.11.001 0896-8411/© 2014 Elsevier Ltd. All rights reserved. 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112 D. Ben-Ami Shor et al. / Journal of Autoimmunity 56 (2015) 111e117 I diabetes mellitus (T1DM) [11e13], and IBD [5,14e18]. Since the in individually ventilated cages, under specific pathogen-free (SPF) “inflammatory bowel disease hygiene hypothesis” [19] has risen, environment. All experiments were approved and executed ac- many groups have investigated the effect of helminthes on colitis, cording to the protocols of the Ethical Committee of the Israeli and different helminthes were shown to suppress colon inflamma- Ministry of Health no.696/11. tion by modulating several immune pathways [18,20e24]. Clinical trials using helminthes to treat IBD were conducted by Weinstock 2.3. Induction of colitis and protocol of treatment et al. [15,16]: ulcerative colitis (UC) and Crohn's disease (CD) patients were treated with ingestion of live Trichuris suis ova (TSO), resulting Colitis was chemically induced by 2% dextransulfate sodium salt in a significant clinical improvement in nearly 80% of the patients (DSS) reagent grade MW-36,000e50,000 (MP Biomedicals with CD and a more modest effect in patients with UC [15,16,25]. To Eschwege, Germany) in tap water and given to the mice for 5 days. date, in all clinical trials conducted, adverse events associated with Three groups of mice were employed in each experiment: 1) DSS helminthic treatment were uncommon, suggesting relative safety [15,16,26]. induced colitis mice treated with TPC (50 mg/0.1 ml PBS) by oral Many of the helminthes species employed in experimental ani- ingestion for 11 days using a feeding needle, beginning at day -2 mal models were shown to limit the inflammatory activity in a va- before disease induction. 2) DSS induced colitis mice treated with a riety of immune-modulated diseases [13,18,27,28]. This implies that control PBS as a vehicle for 11 days, starting at day -2. 3) Healthy, the beneficial effect of the helminthes is not specific to any single non-treated mice. Each group comprised 10 mice. helminth species. Filarial nematodes secrete immunomodulatory molecules mainly comprising of phosphorylcholine (PC)-moieties 2.4. Assessment of DSS colitis glycoporteins, into the host environment [28e33]. ES-62 is the most studied one among them. Its immunosuppressive activity was Throughout the experiment, mice health status was monitored attributed to the PC moieties, covalently attached to N-type glycans daily by the determination of weight loss, rectal bleeding, stool [28,31,32]. Other nematodes like Ascaris suum also express the PC- consistency, and survival. Changes of body weight were indicated as immunomodulatory moiety [34]. PC in nature is presented by percentage loss of the baseline body weight. Occult intestinal apoptotic cells, Gram-positive bacterium including Streptococcus bleeding was followed by using the Hemoccult test (SENSA, Beckman pneumonia, Clostridium, Lactococcus, Lactobacillus, and the Gram- counter, USA) while rectal bleeding signs or gross bleeding were negative bacterium Haemophilus influenzae attached directly to observed. The disease activity index (DAI) was calculated by grading sugar residues, generally considered to be N-acetylgalactosamine on a scale of 0e4 the following parameters: change in weight (0, [34e38]. 1%; 1, 1e5%; 2, 5e10%; 3, 10e20%; and 4, >20%), intestinal bleeding (0, negative; 2, hemoccult; 4, gross bleeding), and stool consistency The idea of finding a synthetic product which can mimic the (0, normal; 2, loose stools; 4, diarrhea). Ten days following disease helminthes-immune-modulatory effect have led us to evaluate the induction the mice were sacrificed and the colon was harvested and protective potential of PC based compound on DSS-induced colitis evaluated for colon length and microscopic colonic damage. in mice. Currently, in the field of autoimmunity there is need for a treatment with small molecules which have a minimal side effects 2.5. Histopathology analysis [37]. The PC is a non-immunogenic natural small molecule, there- fore we conjugated it to tuftsin, coined as TPC, using the tuftsin as a The distal portions of the colon were excised and fixed in 4% self-natural adjuvant [36,39]. Tuftsin is a physiological tetrapeptide formalin. The tissue was dehydrated in alcohol (1 h each in 70, 80, (Thr-Lys-Pro-Arg) fraction of the IgG-heavy-chain molecule pro- 90, and 100%) and xylene (three steps, 1 h for each step) and duced by enzymatic cleavage of the Fc-domain of the heavy chain of IgG in the spleen [40]. Our data are an experimental proof-of- embedded in paraffin. The paraffin block was sliced at a 7 mm concept that TPC therapeutic efficacy is associated with preven- tion of murine colitis development manifested by a reduction in thickness, stained with Hematoxylin-Eosin (H&E) and observed weight loss, intestinal bleeding, colonic length, and histological microscopically. Histological damage and inflammation was damage, resulting in both increased and improved survival. This assessed in a blinded fashion by an expert pathologist. The has important implication in suggesting TPC as a synthetic small following manifestations were included in the evaluations: the molecule which mimics helminthes' derivate for treating colitis. extent of inflammation, depth of inflammation and layers involved, distribution of lesions, and nature of mucosal changes. 2. Materials and methods 2.6. Protein isolation and Western blotting 2.1. TPC Colon tissues were suspended in ice-cold lysis buffer containing Tuftsin extended at its C-terminal i.e THR-LYS-PRO-ARG-Gly-Tyr, 50 mM Tris (pH7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, was synthesized manually following solid phase peptide technol- 1 mM EDTA, 1 mM PMSF, 1 mM sodium vanadate, 0.1% protease ogy (GLS peptide synthesis, Shanghai, China). The peptide was inhibitor mixture (SigmaeAldrich L-4391 St Louis, MO, USA) for coupled to diazotized 4-aminophenyphosphoryl chloride to form 30 min on ice and centrifuged at 13,000 rpm for 20 min. Protein an azo bond between the PC (SigmaeAldrich L-4391 St Louis, MO, concentration was defined by BCA Protein Assay Kit (Pierce, USA) and the tyrosine moiety of the spacer employing procedure as Thermo scientific, Rockford, IL, USA). Tissue lysates were boiled for previously described [41]. The conjugate was characterized by mass 5 min in non-reduced sample buffer and applied to electrophoresed spectra and amino acid analysis as well as by HPLC. on 10% SDS-PAGE, and transferred to nitrocellulose membrane. Following blocking with 10% skim milk, the membranes were 2.2. Mice exposed to the following antibodies: goat-anti-IL-10 monoclonal C57BL/6 male mice weighting 25e30 g were purchased from antibodies, goat anti-IL-17A, goat anti-TNFa, goat-anti-IL-1b and Harlan, Israel. The mice were maintained in a conventional animal housing facility at Sheba Medical Center Israel. The mice were kept anti-GAPDH, all purchased from Santa Cruz. Blots were developed using horseradish peroxidase-conjugated secondary antibodies and the proteins were visualized by chemiluminescence (Santa Cruz Biotechnology, Santa Cruz, CA, USA), using Kodak BioMax film. the ECL detection system (Pierce). Downloaded for Anonymous User (n/a) at Sheba Medical Center from ClinicalKey.com by Elsevier on September 30, 2021. For personal use only. No other uses without permission. Copyright ©2021. Elsevier Inc. All rights reserved. 177

D. Ben-Ami Shor et al. / Journal of Autoimmunity 56 (2015) 111e117 113 2.7. Statistical analysis explore TPC activity, we tested the ability of orally administered Data are expressed as a mean. Weight change and DAI score TPC dosage of 50 mg/0.1 ml PBS to ameliorate the colitis that was differences between TPC-treated and control groups were analyzed using repeated measures analysis of variance (ANOVA) followed by induced by giving DSS [2% (wt/v) for 5 days] to the highly suscep- Tukey method, a single step multiple comparison procedure, for tible mouse strain C57BL/6. The effect of oral treatment, starting comparison between groups. Differences in intestinal bleeding and two days before disease induction, as reflected in DAI score is stool consistency were analyzed using KruskaleWallis non- presented in Fig. 1a. Whereas colitis-untreated mice manifested a parametric analysis followed by ManneWhitney test for compari- maximal average DAI score of 2.59, TPC-treated mice exhibited a son between groups. Differences in colon length and cytokine re- considerably lower average DAI score throughout the entire sponses were compared using one-way analysis of variance experiment, achieving a maximal average score of 1.16. On day 8, (ANOVA) followed by Student's t-test for comparison between the average DAI score of surviving colitis-untreated mice reached to groups. P-values <0.05 were considered to be significant. 2.59 while that of TPC-treated mice amounted to 0.85 (p ¼ 0.001). 3.2. TPC activity on the various manifestations of acute DSS colitis 3. Results The effect of oral TPC treatment on weight change is depicted in Fig. 1b. Whereas colitis-untreated mice suffered from extensive 3.1. The effect of TPC treatment on DSS acute colitis as reflected by weight loss starting two days after DSS administration, averaging the DAI score 6.54% by day 8, mice fed daily with TPC not only experienced considerably less weight loss (maximum of 2.1%) but also regained DSS-induced model is a commonly used model to study various weight to complete restoration of their original weight. Intestinal aspects of IBD such as immune mechanisms, pathogenesis, genetic bleeding, another pathological manifestation of DSS colitis, was predisposition and role of microflora in the pathogenesis [42]. To followed using the Hemoccult test as well as by observation of Fig. 1. TPC effect on colitis development in DSS mice. a: DAI score: The levels of DAI score are presented as an average number, in each group of mice (n ¼ 10 per group), treated with tuftsin-phosphorylcholine (TPC) or PBS. DSS mice that were treated with TPC had significantly lower average DAI score in comparison to mice treated with PBS (p ¼ 0.001). b: weight change: The levels of weight change are presented as percentage in comparison to mice weight on day 0, in each group of mice (n ¼ 10 per group), treated with tuftsin- phosphorylcholine (TPC) or PBS. TPC treated mice suffered significantly less weight loss in comparison to mice treated with PBS (p ¼ 0.007). Downloaded for Anonymous User (n/a) at Sheba Medical Center from ClinicalKey.com by Elsevier on September 30, 2021. For personal use only. No other uses without permission. Copyright ©2021. Elsevier Inc. All rights reserved. 178

114 D. Ben-Ami Shor et al. / Journal of Autoimmunity 56 (2015) 111e117 rectal bleeding. The TPC ingestion reduced the intestinal bleeding. 3.3. Histological assessment of the colonic damage in the acute Hence, on day 8, when all of the untreated mice suffered from DSS-induced colitis visible rectal bleeding, only one (10%) of the TPC-treated mice demonstrated visible rectal bleeding, (p < 0.001). On day 8 all TPC- Microscopical assessment of colonic damage in untreated mice, treated mice except one had a normal stool consistency (90%). In eight days after the colitis induction, revealed a severe inflamma- contrast, only one (10%) of the untreated mice demonstrated a tion, with diffused distribution involving mucosa and submucosa, normal stool consistency while all others secreted either loose stool which, in some cases, also extended to all the intestinal layers or diarrhea (p ¼ 0.001). The DSS regimen applied (2% DSS, 5 days) (transmural penetration). It was also associated with a severe led to 20% mortality in untreated animals compared to the survival disruption of the normal architecture, necrosis, cryptitis, and crypt of all TPC-treated mice. An additional macroscopic indicator for the loss (Fig. 3a). Histological assessment of colon sections from the severity of the colitis is the reduction in colon length (Fig. 2). Eight DSS-induced mice treated with TPC harvested on day eight days after DSS induction, a decrease in colonic length of 33% was revealed a conserved structure with only minimal leukocyte in- found in the colitis-untreated mice compared to the naive mice. In filtrations, similar to the normal colonic histology of the control contrast, only 9% reduction was observed in the TPC-treated mice non-colitis mice (Fig. 3b,c). (p < 0.02). 3.4. Cytokines expression in the colon We analyzed in the colon lysates, the expression of the pro- inflammatory cytokines (TNFa, IL-17, IL-1b) and anti- inflammatory cytokine IL-10 by immunoblot. The expression of TNFa, IL-17 and IL-1b was suppressed in the cell lysates taken from TPC-treated mice, suggesting that TPC attenuated the expression of the pro-inflammatory cytokines. Significant increased IL-10 expression was documented, suggesting that TPC enhanced anti- inflammatory cytokine expression in the colon (Fig. 4). 4. Discussion Fig. 2. TPC effect on colon length in DSS mice. The data presented are length (in This study proved the beneficial effect of TPC in preventing co- centimeter) of colon sections from mice treated with TPC as compare to control PBS litis development in a murine model of colitis induced by DSS. TPC mice at day eight following colitis induction (10 days after TPC was first given). TPC treated mice demonstrated significantly less shortening of the colon (p < 0.02). treatment (daily oral administration of 50 mg/0.1 ml PBS per mouse) in the colitis model induced by one DSS cycle resulted in a sub- stantial beneficial effect in all observed manifestations, including weight loss, intestinal bleeding, colonic length, and histological damage, resulting in both increased and improved survival. Our results suggest that the beneficial effect of TPC is associated with a significant stimulation of anti-inflammatory cytokine IL-10 and suppression of pro-inflammatory cytokines IL-17, TNFa and IL-1b. Environmental factors strongly affect the risk to develop IBD [19]. The high frequency of IBD in industrialized countries led to the notion that the high rate of helminthic colonization in less devel- oped countries may be a protective factor [43]. To support this hypothesis, there are numerous studies showing that different species of helminthes modulate the host's immune sys- tem and protect from colitis in animal models of IBD [14,17,18,20e24,44e50]. Based on this evidence and in the search for new therapeutic strategies, helminthes derived products offer promising potentials. Following exposure to helminthes, key changes were proposed in the innate immune system, including modification of dendritic cells (DCs) [51,52]. Toll like receptors (TLRs) and C-type lectin receptors (CLRs) broadly expressed on DCs are the main target of parasites. Hel- minthic products induce production of the anti-inflammatory cytokine i.e. IL-10 by DCs. By controlling TLR ligand induced DC maturation, helminthic products directly generate an anti- inflammatory effect on DCs [51,52]. Most helminthes stimulate the host to selectively produce Th2 cytokines (IL-4, IL-5, IL-9, IL-13) while blocking Th1 cytokine responses (IL-12, IFN- g) [53,54]. This usually leads to strong responses in immunoglobulin E (IgE), mast cell and eosinophil [6,54]. There is an imbalance in the Th1/Th17/ Th2 network upon helminthes infection which results in lower expression of inflammatory cytokines IL-17, IL-6, IL-1b, IFNg, and TNFa, as well as down-regulation of IL-17 gene expression in the colon and mesenteric lymph node [7,9,10,14,55]. IL-22 was found to Downloaded for Anonymous User (n/a) at Sheba Medical Center from ClinicalKey.com by Elsevier on September 30, 2021. For personal use only. No other uses without permission. Copyright ©2021. Elsevier Inc. All rights reserved. 179

D. Ben-Ami Shor et al. / Journal of Autoimmunity 56 (2015) 111e117 115 Fig. 3. Histological analysis for distal colon section. Histological analysis: H&E staining in representative distal colon section from each studied group of mice as well as from healthy control mice that weren't treated with DSS. a: PBS control mice: Histopathology of distal colon section from DSS mice treated with PBS showed massive infiltration of lymphocyts, destruction of the normal structure and cryptitis b: TPC treated mice: Histopathology of distal colon sections from mice treated with TPC demonstrated normal crypts structure with only minimal cell infiltration c: Healthy non-colitis mice: Histopathology of distal colon sections from healthy mice demonstrated normal crypts structure with only minimal cell infiltration, similar to colon sections from TPC treated mice. have proinflammatory and anti-inflammatory properties at suggested as the culprit of mucositis onset, inducing mucosal bar- different stages of collagen induced arthritis treated with ES 62 [33]. In order to survive in the host environment, helminthes rier breach. Antibody neutralization of IL-1b prevents epithelial upregulated the production of host anti-inflammatory cytokines tight junction dysfunction and alleviates mucositis in mice [65,66]. such as IL-10 and TGFb [33]. Helminthes induce expansion of Treg After treating mice with TPC we observed a decrease in colonic IL- cells subset CD25 þ CD4þ FoxP3þ [8,12]. 1b level, which might represent another pathway for the healing IL-10 is a key anti-inflammatory cytokine that inhibits pro- benefits of TPC. inflammatory responses of both innate and adaptive immune Within their host, helminthes secrete a number of proteins, cells, and is reported to suppress Th1 autoimmune diseases such as T1DM and MS [56,57], as well as aggravate autoantibody-mediated some of which contain phosphorylcholine (PC). Previous evidence autoimmune diseases such as lupus and myasthenia gravis [58,59]. suggests that the protective effect of some helminthes is mediated IL-10 and IL10 receptor (IL10R) deficiencies typically are being by their PC content, which contributes to the immune-modulatory observed within the first year of life with intractable therapy properties of helminthes on lymphocyte responsiveness and cyto- resistant IBD and severe perianal disease [60]. IL10 and IL10R- kine production [28e30,53,67e70]. PC can be found in a wide range deficient mice develop spontaneous intestinal inflammation [61]. of bacterial infections, like Streptococcus pneumonia [71] and Our results confirm the importance of IL-10 in the therapeutic ef- Haemophilus influenza [72]. Examination of the literature reveals fect of TPC, supported by studies indicating that the increased that PC play a role in the regulation of adherence to cells, production of IL-10 is involved in parasite-mediated amelioration encountering the host's innate immune system and then the adaptive immune system and in the regulation of cell growth [38]. of autoimmune diseases [8,12,62e64]. Moreover, IL-1b was It is known, for example, that several human tumors contain elevated levels of PC [73,74] and that ras-transformed cell lines Fig. 4. Colonic cytokines expression following TPC treatment. Ex-vivo analysis of the produce elevated levels of PC, which are necessary for cell prolif- levels of pro-inflammatory cytokines TNFa, IL-17 and IL-1b and anti-inflammatory eration [75,76]. PC is an intermediate in the synthesis of phos- cytokine IL-10 in colon lysates originated from TPC and PBS treated mice were quan- phatidylcholine that is made in a reaction, catalyzed by choline tified by immunoblot. We used glyceraldehyde 3-phosphate dehydrogenase (GAPDH) kinase, which converts ATP and choline into phosphocholine and as loading control. ADP. Phosphatidylcholine is an essential component of the mucus, which is an element of the mucosal surface. It was shown that the mucus phosphatidylcholine content in UC is significantly reduced as compared to controls [77], even when the patients were in remission [78]. Based on these observations, several clinical trials report that oral delayed-release phosphatidylcholine preparation leads to a significant clinical as well as endoscopic improvement in active UC [79e83]. Our aim is to develop a drug for prevention and treatment of autoimmune diseases based on PC. TPC is a new small molecule consisting of a PC conjugated to a self-adjuvant. We chose to use tuftsin, considered to be a “natural adjuvant”, on the basis of its biological functions which encompasses stimulation of phagocytic activity [40,84,85] as well as other stimulatory effects, including Downloaded for Anonymous User (n/a) at Sheba Medical Center from ClinicalKey.com by Elsevier on September 30, 2021. For personal use only. No other uses without permission. Copyright ©2021. Elsevier Inc. All rights reserved. 180

116 D. Ben-Ami Shor et al. / Journal of Autoimmunity 56 (2015) 111e117 increased migration, activation of macrophages and enhance [11] Cooke A. Review series on helminths, immune modulation and the hygiene chemotaxis of monocytes [40,84e86]. Tuftsin in a branched pep- hypothesis: how might infection modulate the onset of type 1 diabetes? tide form was used as a natural adjuvant for developing vaccine to Immunology 2009;126:12e7. influenza with no side effects [39]. TPC is being developed as a subcutaneous and oral treatment. While the drug is expected to [12] Zaccone P, Burton OT, Gibbs S, Miller N, Jones FM, Dunne DW, et al. Immune serve for treatment and vaccination for diverse autoimmune con- modulation by schistosoma mansoni antigens in NOD mice: effects on both ditions, we present here the results of the therapeutic potential of innate and adaptive immune systems. J Biomed Biotechnol 2010;2010: TPC in DSS-induced model of colitis. So far, in small clinical trials 795210. the ova treatments appear to be successful and safe for IBD patients [15,16,26], and the idea of using TPC molecule is promising as it is a [13] Cooke A, Tonks P, Jones FM, O'Shea H, Hutchings P, Fulford AJ, et al. Infection synthetic analog of the natural products. with schistosoma mansoni prevents insulin dependent diabetes mellitus in non-obese diabetic mice. Parasite Immunol 1999;21:169e76. In conclusion, in the presented study we showed that TPC treatment can prevent severe experimental colitis development in [14] Ruyssers NE, De Winter BY, De Man JG, Loukas A, Pearson MS, Weinstock JV, naïve mice, as assessed by weight loss, colon length, and disease et al. Therapeutic potential of helminth soluble proteins in TNBS-induced activity score as well as histological analysis. We believe that our colitis in mice. Inflamm Bowel Dis 2009;15:491e500. data has a substantial implication in suggesting TPC as a synthetic small molecule which mimics helminthes' derivate for treating [15] Summers RW, Elliott DE, Urban Jr JF, Thompson R, Weinstock JV. Trichuris suis colitis. However, one must keep in mind that this experiment used therapy in Crohn's disease. Gut 2005;54:87e90. chemical induced colitis model which does not fully mimic the immune phenomena of IBD in humans in which multiple immune [16] Summers RW, Elliott DE, Urban Jr JF, Thompson RA, Weinstock JV. Trichuris pathologies may intertwine. Moreover, this is a preventive study suis therapy for active ulcerative colitis: a randomized controlled trial. and future studies must focus on models with existing chronic Gastroenterology 2005;128:825e32. disease. [17] Elliott DE, Li J, Blum A, Metwali A, Qadir K, Urban Jr JF, et al. Exposure to Author contribution schistosome eggs protects mice from TNBS-induced colitis. Am J Physiol Gastrointest Liver Physiol 2003;284:G385e91. All authors were involved in drafting the article and approved the final version to be published. Study conception and design: Dr. [18] Hang L, Setiawan T, Blum AM, Urban J, Stoyanoff K, Arihiro S, et al. Heli- Shoenfeld, Dr. Blank and Dr. Ben-Ami Shor. Experimental work was gmosomoides polygyrus infection can inhibit colitis through direct interaction done by Dr. Blank and Dr. Ben-Ami Shor. Dr. Fridkin constructed the with innate immunity. J Immunol 2010;185:3184e9. TPC molecule. Bashi, Lachnish and Bizzaro took part in performing the study. Dr. Barshak covered the histological part. [19] Weinstock JV, Elliott DE. Helminths and the IBD hygiene hypothesis. Inflamm Bowel Dis 2009;15:128e33. Acknowledgment [20] Khan WI, Blennerhasset PA, Varghese AK, Chowdhury SK, Omsted P, Deng Y, This study has partially supported by TEVA Company and et al. 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Gut 2004;53:99e107. immune mechanisms in helminth infection. Nat Rev Immunol 2007;7: 975e87. [25] Elliott DE, Weinstock JV. Helminthic therapy: using worms to treat immune- mediated disease. Adv Exp Med Biol 2009;666:157e66. [5] Kuijk LM, van Die I. Worms to the rescue: can worm glycans protect from autoimmune diseases? IUBMB Life 2010;62:303e12. [26] Sandborn WJ, Elliott DE, Weinstock J, Summers RW, Landry-Wheeler A, Silver N, et al. Randomised clinical trial: the safety and tolerability of Trichuris [6] Ben-Ami Shor D, Harel M, Eliakim R, Shoenfeld Y. The hygiene theory har- suis ova in patients with Crohn's disease. Alimentary Pharmacol Ther nessing helminths and their ova to treat autoimmunity. Clin Rev Allergy 2013;38:255e63. Immunol 2013;45:211e6. [27] Correale J, Farez MF. The impact of parasite infections on the course of mul- [7] Wu Z, Nagano I, Asano K, Takahashi Y. Infection of non-encapsulated species tiple sclerosis. J Neuroimmunol 2011;233:6e11. of trichinella ameliorates experimental autoimmune encephalomyelitis involving suppression of Th17 and Th1 response. Parasitol Res 2010;107: [28] Harnett MM, Kean DE, Boitelle A, McGuiness S, Thalhamer T, Steiger CN, et al. 1173e88. The phosphorycholine moiety of the filarial nematode immunomodulator ES- 62 is responsible for its anti-inflammatory action in arthritis. Ann Rheum Dis [8] Correale J, Farez M. Association between parasite infection and immune re- 2008;67:518e23. sponses in multiple sclerosis. Ann Neurol 2007;61:97e108. [29] McInnes IB, Leung BP, Harnett M, Gracie JA, Liew FY, Harnett W. A novel [9] Osada Y, Shimizu S, Kumagai T, Yamada S, Kanazawa T. Schistosoma mansoni therapeutic approach targeting articular inflammation using the filarial infection reduces severity of collagen-induced arthritis via down-regulation of nematode-derived phosphorylcholine-containing glycoprotein ES-62. pro-inflammatory mediators. Int J Parasitol 2009;39:457e64. J Immunol 2003;171:2127e33. [10] Pineda MA, McGrath MA, Smith PC, Al-Riyami L, Rzepecka J, Gracie JA, et al. [30] Harnett W, Harnett MM. Phosphorylcholine: friend or foe of the immune The parasitic helminth product ES-62 suppresses pathogenesis in collagen- system? Immunol Today 1999;20:125e9. induced arthritis by targeting the interleukin-17-producing cellular network at multiple sites. Arthritis Rheum 2012;64:3168e78. [31] Goodridge HS, McGuiness S, Houston KM, Egan CA, Al-Riyami L, Alcocer MJ, et al. Phosphorylcholine mimics the effects of ES-62 on macrophages and dendritic cells. Parasite Immunol 2007;29:127e37. [32] Harnett W, Harnett MM. Immunomodulatory activity and therapeutic po- tential of the filarial nematode secreted product, ES-62. Adv Exp Med Biol 2009;666:88e94. [33] Pineda MA, Rodgers DT, Al-Riyami L, Harnett W, Harnett M. ES-62 protects against collagen-induced arthritis by resetting IL-22 towards resolution of inflammation in the joints. Arthritis Rheumatol 2014;66(6):1492e503. [34] Poltl G, Kerner D, Paschinger K, Wilson IB. N-glycans of the porcine nematode parasite Ascaris suum are modified with phosphorylcholine and core fucose residues. FEBS J 2007;274:714e26. [35] Tomasz A. Choline in the cell wall of a bacterium: novel type of polymer- linked choline in Pneumococcus. Science 1967;157:694e7. [36] Peng Y, Martin DA, Kenkel J, Zhang K, Ogden CA, Elkon KB. Innate and adaptive immune response to apoptotic cells. J Autoimmun 2007;29:303e9. [37] Shor DB, Shoenfeld Y. Autoimmunity: will worms cure rheumatoid arthritis? Nat Rev Rheumatol 2013;9:138e40. [38] Young NM, Foote SJ, Wakarchuk WW. Review of phosphocholine substituents on bacterial pathogen glycans: synthesis, structures and interactions with host proteins. Mol Immunol 2013;56:563e73. [39] Liu X, Guo J, Han S, Yao L, Chen A, Yang Q, et al. 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D. Ben-Ami Shor et al. / Journal of Autoimmunity 56 (2015) 111e117 117 [40] Najjar VA, Nishioka K. “Tuftsin”: a natural phagocytosis stimulating peptide. [65] Kanarek N, Grivennikov SI, Leshets M, Lasry A, Alkalay I, Horwitz E, et al. Nature 1970;228:672e3. Critical role for IL-1beta in DNA damage-induced mucositis. Proc Natl Acad Sci U S A 2014;111:E702e11. [41] Koch Y, Wilchek M, Fridkin M, Chobsieng P, Zor U, Lindner HR. Production and characterization of an antiserum to synthetic gonadotropin-releasing hor- [66] Al-Sadi RM, Ma TY. IL-1beta causes an increase in intestinal epithelial tight mone. Biochem Biophysical Res Commun 1973;55:616e22. junction permeability. J Immunol 2007;178:4641e9. [42] Perse M, Cerar A. Dextran sodium sulphate colitis mouse model: traps and [67] Rzepecka J, Coates ML, Saggar M, Al-Riyami L, Coltherd J, Tay HK, et al. Small tricks. J Biomed Biotechnol 2012;2012:718617. molecule analogues of the immunomodulatory parasitic helminth product ES- 62 have anti-allergy properties. Int J Parasitol 2014;44:669e74. [43] Summers RW, Elliott DE, Weinstock JV. Is there a role for helminths in the therapy of inflammatory bowel disease? Nat Clin Pract Gastroenterol Hepatol [68] Al-Riyami L, Pineda MA, Rzepecka J, Huggan JK, Khalaf AI, Suckling CJ, et al. 2005;2:62e3. Designing anti-inflammatory drugs from parasitic worms: a synthetic small molecule analogue of the Acanthocheilonema viteae product ES-62 prevents [44] Reardon C, Sanchez A, Hogaboam CM, McKay DM. Tapeworm infection re- development of collagen-induced arthritis. J Med Chem 2013;56: duces epithelial ion transport abnormalities in murine dextran sulfate 9982e10002. sodium-induced colitis. Infect Immun 2001;69:4417e23. [69] Houston KM, Harnett W. Mechanisms underlying the transfer of phosphor- [45] Elliott DE, Urban JJ, Argo CK, Weinstock JV. Does the failure to acquire hel- ylcholine to filarial nematode glycoproteinsea possible role for choline ki- minthic parasites predispose to Crohn's disease? FASEB J 2000;14:1848e55. nase. Parasitology 1999;118(Pt 3):311e8. [46] Mo HM, Liu WQ, Lei JH, Cheng YL, Wang CZ, Li YL. Suppression of Schistosoma [70] Lal RB, Ottesen EA. Phosphocholine epitopes on helminth and protozoal japonicum eggs on TNBS-induced colitis in mice. Zhongguo Ji Sheng Chong parasites and their presence in the circulation of infected human patients. Xue Yu Ji Sheng Chong Bing Za Zhi 2007;25:381e4. Trans R Soc Trop Med Hyg 1989;83:652e5. [47] Jiang J, Xue RY, Zhang SC, Zhou J, Zhou K. [Effect of schistosome ova on tri- [71] Mukerji R, Mirza S, Roche AM, Widener RW, Croney CM, Rhee DK, et al. nitrobenzenesulfonic acid induced colitis in mice]. Zhonghua Yi Xue Za Zhi Pneumococcal surface protein A inhibits complement deposition on the 2007;87:2149e51. pneumococcal surface by competing with the binding of C-reactive protein to cell-surface phosphocholine. J Immunol 2012;189:5327e35. [48] Mo HM, Liu WQ, Lei JH, Cheng YL, Wang CZ, Li YL. Schistosoma japonicum eggs modulate the activity of CD4þ CD25þ Tregs and prevent development of [72] Schweda EK, Brisson JR, Alvelius G, Martin A, Weiser JN, Hood DW, et al. colitis in mice. Exp Parasitol 2007;116:385e9. Characterization of the phosphocholine-substituted oligosaccharide in lipo- polysaccharides of type b Haemophilus influenzae. Eur J Biochem/FEBS [49] Motomura Y, Wang H, Deng Y, El-Sharkawy RT, Verdu EF, Khan WI. Helminth 2000;267:3902e13. antigen-based strategy to ameliorate inflammation in an experimental model of colitis. Clin Exp Immunol 2009;155:88e95. [73] Daly PF, Lyon RC, Faustino PJ, Cohen JS. Phospholipid metabolism in cancer cells monitored by 31P NMR spectroscopy. J Biological Chem 1987;262: [50] Weinstock GM. Genomic approaches to studying the human microbiota. Na- 14875e8. ture 2012;489:250e6. [74] Miceli MV, Kan LS, Newsome DA. Phosphorus-31 nuclear magnetic resonance [51] Kane CM, Cervi L, Sun J, McKee AS, Masek KS, Shapira S, et al. Helminth an- spectroscopy of human retinoblastoma cells: correlation with metabolic tigens modulate TLR-initiated dendritic cell activation. J Immunol 2004;173: indices. Biochimica Biophysica Acta 1988;970:262e9. 7454e61. [75] Hernandez-Alcoceba R, Saniger L, Campos J, Nunez MC, Khaless F, Gallo MA, [52] Duez C, Gosset P, Tonnel AB. Dendritic cells and toll-like receptors in allergy et al. Choline kinase inhibitors as a novel approach for antiproliferative drug and asthma. Eur J Dermatol 2006;16:12e6. design. Oncogene 1997;15:2289e301. [53] Al-Riyami L, Harnett W. Immunomodulatory properties of ES-62, a [76] Ratnam S, Kent C. Early increase in choline kinase activity upon induction of phosphorylcholine-containing glycoprotein secreted by acanthocheilonema the H-ras oncogene in mouse fibroblast cell lines. Archives Biochem viteae. Endocr Metabolic Immune Disord Drug Targets 2012;12:45e52. Biophysics 1995;323:313e22. [54] Gause WC, Urban Jr JF, Stadecker MJ. The immune response to parasitic hel- [77] Ehehalt R, Wagenblast J, Erben G, Lehmann WD, Hinz U, Merle U, et al. minths: insights from murine models. Trends Immunol 2003;24:269e77. Phosphatidylcholine and lysophosphatidylcholine in intestinal mucus of ul- cerative colitis patients. A quantitative approach by nanoElectrospray-tandem [55] Harnett MM, Melendez AJ, Harnett W. The therapeutic potential of the filarial mass spectrometry. Scand J Gastroenterology 2004;39:737e42. nematode-derived immunodulator, ES-62 in inflammatory disease. Clin Exp Immunol 2010;159:256e67. [78] Braun A, Treede I, Gotthardt D, Tietje A, Zahn A, Ruhwald R, et al. Alterations of phospholipid concentration and species composition of the intestinal [56] Pennline KJ, Roque-Gaffney E, Monahan M. Recombinant human IL-10 pre- mucus barrier in ulcerative colitis: a clue to pathogenesis. Inflamm Bowel Dis vents the onset of diabetes in the nonobese diabetic mouse. Clin Immunol 2009;15:1705e20. Immunopathol 1994;71:169e75. [79] Stremmel W, Merle U, Zahn A, Autschbach F, Hinz U, Ehehalt R. Retarded [57] Rott O, Fleischer B, Cash E. Interleukin-10 prevents experimental allergic release phosphatidylcholine benefits patients with chronic active ulcerative encephalomyelitis in rats. Eur J Immunol 1994;24:1434e40. colitis. Gut 2005;54:966e71. [58] Zhang GX, Xiao BG, Yu LY, van der Meide PH, Link H. Interleukin 10 aggravates [80] Stremmel W, Braun A, Hanemann A, Ehehalt R, Autschbach F, Karner M. experimental autoimmune myasthenia gravis through inducing Th2 and B cell responses to AChR. J Neuroimmunol 2001;113:10e8. Delayed release phosphatidylcholine in chronic-active ulcerative colitis: a randomized, double-blinded, dose finding study. J Clin gastroenterology [59] Ishida H, Muchamuel T, Sakaguchi S, Andrade S, Menon S, Howard M. 2010;44:e101e7. Continuous administration of anti-interleukin 10 antibodies delays onset of [81] Stremmel W, Gauss A. Lecithin as a therapeutic agent in ulcerative colitis. Dig autoimmunity in NZB/W F1 mice. J Exp Med 1994;179:305e10. Dis 2013;31:388e90. [82] Stremmel W, Hanemann A, Ehehalt R, Karner M, Braun A. Phosphatidylcholine [60] Shah N, Kammermeier J, Elawad M, Glocker EO. Interleukin-10 and inter- (lecithin) and the mucus layer: evidence of therapeutic efficacy in ulcerative leukin-10-receptor defects in inflammatory bowel disease. Curr Allergy colitis? Dig Dis 2010;28:490e6. Asthma Reports 2012;12:373e9. [83] Karner M, Kocjan A, Stein J, Schreiber S, von Boyen G, Uebel P, et al. First multicenter study of modified release phosphatidylcholine “LT-02” in ulcer- [61] Shouval DS, Ouahed J, Biswas A, Goettel JA, Horwitz BH, Klein C, et al. Inter- ative colitis: a randomized, placebo-controlled trial in mesalazine-refractory leukin 10 receptor signaling: master regulator of intestinal mucosal homeo- courses. Am J Gastroenterology 2014;109:1041e51. stasis in mice and humans. Adv Immunol 2014;122:177e210. [84] Siemion IZ, Kluczyk A. Tuftsin: on the 30-year anniversary of Victor Najjar's discovery. Peptides 1999;20:645e74. [62] Elliott DE, Weinstock JV. Helminth-host immunological interactions: pre- [85] Fridkin M, Najjar VA. Tuftsin: its chemistry, biology, and clinical potential. Crit vention and control of immune-mediated diseases. Ann N. Y Acad Sci Rev Biochem Mol Biol 1989;24:1e40. 2012;1247:83e96. [86] Lukacs K, Szabo G, Sonkoly I, Vegh E, Gacs J, Szekerke M, et al. Stimulating effect of tuftsin and its analogues on the defective monocyte chemotaxis in [63] Zaccone P, Fehervari Z, Jones FM, Sidobre S, Kronenberg M, Dunne DW, et al. systemic lupus erythematosus. Immunopharmacology 1984;7:171e8. Schistosoma mansoni antigens modulate the activity of the innate immune response and prevent onset of type 1 diabetes. Eur J Immunol 2003;33: 1439e49. [64] Nagayama Y, Watanabe K, Niwa M, McLachlan SM, Rapoport B. Schistosoma mansoni and alpha-galactosylceramide: prophylactic effect of Th1 immune suppression in a mouse model of Graves' hyperthyroidism. J Immunol 2004;173:2167e73. Downloaded for Anonymous User (n/a) at Sheba Medical Center from ClinicalKey.com by Elsevier on September 30, 2021. For personal use only. No other uses without permission. Copyright ©2021. Elsevier Inc. All rights reserved. 182

‫‪The many etiologies of neonatal hypocalcemic seizures‬‬ ‫‪Pediatric Emergency Care | 2015‬‬ ‫במהלך השנים זכיתי להנחות ארבע סטודנטיות מוכשרות במסגרת פרויקט‬ ‫ח\"ץ‪ .‬העבודה עימן היתה מפרה ומתגמלת באופן הדדי‪ ,‬וניתן להתרשם‬ ‫מתוצאותיה בחוברת זו‪ .‬אני משוכנעת שכל אחת מהן תצליח בהמשך דרכה‪.‬‬ ‫אני רואה בהדרכה והוראה נדבך חשוב בתפקיד שלנו כרופאים‪ ,‬ומלאת‬ ‫הערכה לפרויקט על החיבור המוצלח עם חוקרים צעירים‪.‬‬ ‫מנחה‪ :‬יעל לוי‪-‬שרגא‬ ‫אנדוקרינולוגיה ילדים‬ ‫‪[email protected]‬‬ ‫‪Keren Dallalzadeh‬‬ ‫מנחה‪ :‬פרופ' אורית חמיאל‬ ‫אונ' תל אביב תכנית ניו יורק‬ ‫השתתפה כסטודנטית בפרויקט ח“ץ‬ ‫מנהלת יחידה לאנדוקרינולוגיה ילדים‬ ‫בין השנים ‪2012-2013‬‬ ‫‪[email protected]‬‬ ‫‪[email protected]‬‬ ‫‪183‬‬

ILLUSTRATIVE CASES The Many Etiologies of Neonatal Hypocalcemic Seizures Yael Levy-Shraga, MD,*† Keren Dallalzadeh, MD,† Keren Stern, MD,†‡ Gideon Paret, MD,†‡ and Orit Pinhas-Hamiel, MD† Abstract: Seizures during the neonatal period have a broad differential di- jerking, generalized or focal seizures,6,7 stridor (secondary to agnosis. Unlike in developing countries where hypovitaminosis D and hypo- laryngospasm),8 wheezing (secondary to bronchospasm), vo- calcemia constitutes a major cause of infantile seizures, the number of miting (secondary to pylorospasm), and prolonged QTc on elec- neonatal seizures attributed to hypocalcemia in developed countries has de- trocardiogram.9 Cardiac function may also be impaired because creased dramatically due to the improvement of infant formulas and vitamin of poor muscle contractility.10 The severity of these symptoms D supplementation. In these countries, most infants that present with hypo- may range from very mild to life threatening.11 calcemic seizures have underlying endocrinological etiologies rather than di- etary insufficiencies. Here, we describe 3 cases of neonatal seizures due to This case series discusses 3 examples of severe neonatal hy- hypocalcemia. Although the symptoms and calcium concentrations at pre- pocalcemia, each with a complex course of disease. Although the sentation were similar in all 3 cases, the course of the disease and the final clinical presentations were similar, the etiologies varied, and they diagnosis for each were distinct. The cases are presented along with a brief therefore shed light on the complex differential diagnosis of hypo- review of the pathophysiology, differential diagnosis, and treatment of calcemia in neonates. neonatal hypocalcemia. CASE 1 Key Words: neonatal hypocalcemia, seizures, vitamin D A 1-month-old previously healthy female infant presented to (Pediatr Emer Care 2015;31: 197–201) the pediatric emergency department after 4 seizure episodes 2 days before admission. The episodes included tonic-clonic movements N eonatal hypocalcemia is a potentially life-threatening condi- of all 4 limbs for about 30 seconds followed by a postictal state. tion, with reported prevalence varying by gestational age, maternal and infant comorbidities, and perinatal factors.1 It is de- The patient was conceived by in vitro fertilization through an fined as a total serum calcium concentration of less than 8 mg/dL anonymous egg donor. She was delivered via cesarean delivery at (2 mmol/L) or ionized calcium level less than 4.8 mg/dL 38 weeks gestation after a pregnancy complicated by gestational dia- (1.2 mmol/L) in term infants and total calcium level less than betes and preeclampsia. Birth weight was 2.830 kg with Apgar scores 7 mg/dL (1.75 mmol/L) or ionized calcium 4 mg/dL (1 mmol/L) of 9 and 10 at 1 and 5 minutes, respectively. There were no postnatal in premature infants.2 complications; however, the parents described irritability and jitter- iness soon after birth. The patient was fed with infant formula. Calcium homeostasis begins in utero. Active calcium trans- fer from maternal circulation to the fetus occurs via a transplacen- Physical examination revealed an active, afebrile baby with tal calcium pump regulated by parathyroid hormone (PTH)– normal facial appearance. Laboratory investigation included com- related peptide, primarily during the third trimester of pregnancy. plete blood cell count, urinalysis, and cerebrospinal fluid analysis This process results in a higher plasma calcium concentration in that were within reference range. Blood chemistry revealed calcium the fetus compared with that of the mother and leads to fetal hy- level of 5.8 mg/dL (reference range, 8–10.5 mg/dL) and phospho- percalcemia at term, with total calcium concentrations in umbili- rus level of 11.1 mg/dL (reference range, 4.5–6.7; Table 1). The cal cord blood reaching 10 to 11 mg/dL (2.5–2.75 mmol/L).3 patient was started on intravenous (IV) 10% calcium gluconate Placental transfer stops abruptly after birth. Within the first few according to the protocol for management of severe hypocalce- hours of life, serum calcium concentrations begin to fall and reach mia.4 Endocrine function testing showed a serum intact PTH a trough value by the second or third day of life.3 The calcium (iPTH) level of 13.1 pg/mL (reference range, 16–87 pg/mL), inap- level then increases to normal values seen in children and adults propriately low for her hypocalcemia, thyroid stimulating hor- by the tenth day of life. This homeostasis after birth depends on mone of 2.38 mIU/L (reference range, 0.7–5.9 mIU/L), and free PTH secretion, dietary calcium intake, renal calcium reabsorption, thyroxine of 13.8 pmol/L (reference range, 7–16 pmol/L). skeletal calcium stores, and vitamin D levels.4 The patient was admitted to the pediatric intensive care unit A constant extracellular calcium concentration is essential for further treatment and investigation. She was treated with IV for cellular membrane integrity and cellular functioning. Calcium calcium gluconate for 72 hours and was then switched to oral plays a key role in a multitude of biochemical processes as a sec- calcium carbonate (elemental calcium 50 mg/kg per day), ond messenger, serves as a cofactor in blood coagulation, and is a alfacalcidol (alpha D3, 0.5 μg) and low phosphorus formula significant ion in neuromuscular excitability.5 Clinical manifesta- (Similac 60/40) with normalization of serum calcium and phos- tions of neonatal hypocalcemia include jitteriness, tetany, muscle phorus levels. Upon further investigation, kidney ultrasound re- vealed bilateral hydronephrosis. Chest x-ray, echocardiography, From the *Pediatric Endocrine and Diabetes Unit, Safra Children's Hospital, brain ultrasound, and electroencephalography revealed no abnormal- Sheba Medical Center, Ramat Gan; †Sackler School of Medicine, Tel-Aviv Uni- ities. Fluorescent in situ hybridization testing for a microdeletion on versity, Tel-Aviv; and ‡Department of Pediatric Intensive Care, Safra Children's chromosome 22q11 was negative, ruling out DiGeorge syndrome. Hospital, Sheba Medical Center, Ramat Gan, Israel. Disclosure: The authors declare no conflict of interest. At follow-up visits, the treatment was tapered down gradu- Reprints: Yael Levy-Shraga, MD, Pediatric Endocrine and Diabetes Unit, Safra ally until cessation at 10 months of age. The patient was well and normocalcemic after discontinuation of medication. Children's Hospital, Sheba Medical Center, Tel Hashomer 52621, Ramat Gan, Israel (e‐mail: [email protected]). After further investigation, the patient's mother's blood test Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. showed calcium level of 10.2 mg/dL (reference range, 8.4– ISSN: 0749-5161 10.2 mg/dL), phosphorus level of 3 mg/dL (reference range, 2.8–4.6 mg/dL), iPTH level of 100 pg/mL (reference range, 10– Pediatric Emergency Care • Volume 31, Number 3, March 2015 65 pg/mL), and 25-hydroxy-vitamin D level of 17 ng/mL www.pec-online.com 197 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. 184

Levy-Shraga et al Pediatric Emergency Care • Volume 31, Number 3, March 2015 TABLE 1. Biochemical Data at Presentation Albumin, g/dL Case 1 Case 2 Case 3 Reference Range Calcium, total, mg/dL Calcium, ionized, mmol/L 3.6 3.7 3.1 3.4–5.0 Phosphorus, mg/dL 5.8 6.0 6.0 8.5–10.5 Magnesium, mg/dL 0.69 0.66 0.68 PTH, intact, pg/mL 11.1 6.7 10.6 1–1.3 25-hydroxy-vitamin D, ng/mL 2.1 1.7 1.4 4.5–6.7 1,25-dihydroxyvitamin D, pg/mL 13.1 300 10 1.7–2.4 Alkaline phosphatase, IU/L 34 4.5 31 16–87 ND 30 ND 30–100 ND, not done. 351 1214 235 20–100 145–320 (reference range, 30–100 ng/mL). Because the elevated iPTH of persistent mildly elevated liver enzymes, a liver biopsy was done level can be attributed to vitamin D insufficiency, recurrent blood that revealed marked steatosis and portal fibrosis with early septal for- tests were conducted after vitamin D supplementation and re- mation. The etiology for the fatty liver still remains unknown. vealed maternal calcium level of 10.7 mg/dL, phosphorus level of 2.4 mg/dL, 25-hydroxy-vitamin D level of 28 ng/mL, and iPTH The diagnoses include (1) vitamin D deficiency and level of 118 pg/mL. (2) hepatic steatosis. The diagnosis was transient hypoparathyroidism due to TABLE 2. Differential Diagnosis of Early- and Late-Onset maternal hyperparathyroidism. Neonatal Hypocalcemia CASE 2 Early-Onset Hypocalcemia • Prematurity A 2-month-old male infant presented to the pediatrics emer- • Intrauterine growth retardation gency department due to jittering in all 4 limbs since birth. The • Preeclampsia patient's parents and siblings were Sephardic Jews of Moroccan • Perinatal stress/asphyxia ancestry and healthy. The patient was fed with infant formula • Maternal intake of anticonvulsants and was given 400 IU/d of cholecalciferol (vitamin D3) as needed. • Maternal hyperparathyroidism • Maternal diabetes Physical and neurological examination results were normal. • Hyperbilirubinemia Complete blood cell count was unremarkable. Blood chemistry re- • Drugs: bicarbonate, phosphate, citrate (present in transfused blood), vealed a serum calcium level of 6.0 mg/dL, phosphorus level of fatty acid, aminoglycosides 6.7 mg/dL, and alkaline phosphatase level of 1214 (reference range, 145–320 IU/L; Table 1). Bilirubin, liver enzyme levels, and kidney Late-Onset Hypocalcemia function, as well as complete blood cell count, were all normal. The • Hypoparathyroidism patient was treated with IV 10% calcium gluconate. 1. Transient neonatal 2. Dysgenesis/agenesis of the parathyroid glands During his hospitalization in the intensive care unit, the patient's a. Isolated hypoparathyroidism (GCM2, PTH, SOX3) calcium level remained low (ionized calcium level, 0.84 mmol/L) b. DiGeorge syndrome (22q11.2 deletion) with mild lactic acidosis (pH 7.313; PCO2, 36 mm Hg; bicarbonate c. Sanjad-Sakati syndrome (TCBE) level, 18 mmol/L; lactate level, 21 mg/dL). The patient's iPTH level d. Barakat syndrome (GATA3) was 300 pg/mL (reference range, 10–65 pg/mL), and 25-hydroxy- e. Mitochondrial disorders (Kearns-Sayre, Pearson, vitamin D was 4.5 ng/mL (reference range, 30–100 ng/mL). mitochondrial encephalopathy, lactic acidosis, stroke-like episodes) Upon further investigation, ECG showed a long QTc interval 3. Dyshormonogenesis (0.43 seconds). X-ray of the right femur raised a suspicion of rick- 4. Hypercalciuric hypocalcemia (CASR) ets. The level of 1,25-dihydroxyvitamin D was 30 pg/mL (refer- • Insensitivity to PTH ence range, 20–100 pg/mL), urine calcium/creatinine ratio was 1. Blomstard chondrodysplasia (PTHR) 0.12, and total reabsorption of phosphorus was 70.7%. His 2. Pseudohypoparathyroidism (type IA [GNAS], IB, IC, II) mother's blood test revealed normal levels of calcium, phospho- • Vitamin D deficiency rus, and PTH. Her level of 25-hydroxy-vitamin D was 21 ng/mL. Nutritional, malabsorption, maternal vitamin D deficiency, hepatobiliary disease, renal failure, and impaired The patient required prolonged therapy with calcium supple- vitamin D metabolism mentation, cholecalciferol (vitamin D3) 4000 IU/d, and alfacalcidol • Vitamin D resistance 0.5 μg/d due to persistent hypocalcemia. Ten months after admission, • Hypomagnesemia with no medical therapy, the following laboratory results were ob- • Increased phosphate load tained: calcium, 10.1 mg/dL; phosphorus, 6.4 mg/dL; alkaline phos- phatase, 259 IU/L; iPTH, 54 mg/dL; 25-hydroxy-vitamin D, © 2015 Wolters Kluwer Health, Inc. All rights reserved. 36 ng/mL; and 1,25-dihydroxyvitamin D, 97 pg/mL. Interestingly, during his admission in the intensive care unit, there was mild elevation of liver enzymes, with a peak aspartate ami- notransferase level of 123 IU/L (reference range, 0–60 IU/L) and al- anine aminotransferase of 186 IU/L (reference range, 7–45 IU/L). Bilirubin and γ-glutamyltransferase levels were normal. After a year 198 www.pec-online.com Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. 185

Pediatric Emergency Care • Volume 31, Number 3, March 2015 Neonatal Hypocalcemia CASE 3 3. Autistic spectrum disorder–She was diagnosed recently and en- rolled in an early intervention program and speech therapy at a The patient was born at 2.7 kg after 37 weeks gestation child development center. via cesarean delivery and was a fraternal twin. The parents were Ashkenazi Jews of Russian descent, and the patient's parents The diagnosis was DiGeorge syndrome. and 5-year-old sister were all healthy. During the first day of life, 2 episodes of jitteriness and cyanosis were noted. During the sec- DISCUSSION ond episode, oxygen saturation decreased to 82%, and the infant was transferred to the neonatal intensive care unit to begin an ex- Our cases illustrate the various etiologies of neonatal hypo- tensive workup. Upon physical examination, the patient had calcemia. Hypocalcemia has been a reported etiology for neonatal slightly dysmorphic facial features and symmetrically low-set convulsions for 100 years. The first report of neonatal seizures ears. Blood test revealed a total calcium level of 7.5 mg/dL on secondary to hypocalcemia was by Kehrer12 in 1913. Forty years the second day of life that decreased to 6 mg/dL on the fifth day. ago, a retrospective study from Britain found that 92% of newborn Also on the fifth day of life, phosphorus level was 10.6 mg/dL, infants who had seizures secondary to a metabolic abnormality iPTH level was 10 pg/mL, and magnesium level was 1.4 mg/dL were hypocalcemic.13 Since then, the prevalence of hypocalcemic (reference range, 1.7–2.4 mg/dL; Table 1). Fluorescent in situ hy- seizure has decreased dramatically to as low as 3% of the neonatal bridization analysis was positive for a 22q11 deletion, thus con- seizure population, likely as a result of improvement in infant for- firming the diagnosis. The patient was treated with IV calcium mula.14,15 However, in developing countries, hypocalcemic sei- gluconate, and total calcium level was corrected within 48 hours. zures are not unusual.16–18 She was then switched to oral calcium carbonate, alfacalcidol, and low phosphate formula (Similac 60/40). Upon further investiga- Neonatal hypocalcemia is conventionally divided into early tion, echocardiogram revealed a patent foramen ovale and right onset, manifesting within the first 24 to 72 hours of life, and late aortic arch. Immunoglobulin testing showed normal subpopula- onset, occurring between 4 and 28 days of age (Table 2). Transient tions of lymphocytes. early-onset hypocalcemia is common and has multiple etiologies. It may be due to maternal factors such as gestational diabetes, pre- Throughout follow-up care, the following problems were eclampsia, vitamin D deficiency, anticonvulsant use, and maternal encountered: hyperparathyroidism. Early hypocalcemia may also be due to neo- natal factors such as prematurity, intrauterine growth retardation, 1. Hypercalciuria–Urine calcium/creatinine ratio reached a maxi- and birth asphyxia. Neonatal sepsis, respiratory distress syn- mum of 1.8 at the age of 2 months. Calcium carbonate therapy drome, hypomagnesemia, hyperbilirubinemia, renal failure, and was tapered down until cessation at the age of 9 months. Since exogenous or iatrogenic causes may also be the culprits.19 then, she has had normal levels of calcium and phosphorus with- out therapy. Ultrasound of the kidneys showed nephrocalcinosis The etiologies of late neonatal hypocalcemia can be classi- that resolved by the next follow-up examination. fied into 2 groups based on the levels of PTH. Low levels of PTH can be classified by its pathogenesis: an error in the embryo- 2. Chronic serous otitis media with hearing loss. genesis of the parathyroid gland (hypoplasia/aplasia) or a defect in the synthesis or release of PTH (dyshormonogenesis). The FIGURE 1. Management of neonatal hypocalcemia. www.pec-online.com 199 © 2015 Wolters Kluwer Health, Inc. All rights reserved. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. 186

Levy-Shraga et al Pediatric Emergency Care • Volume 31, Number 3, March 2015 hypoparathyroidism may be isolated (GCM2, PTH, SOX3) or as of infants are exclusive prolonged breast-feeding18 and lack of part of a syndrome (DiGeorge syndrome, Barakat syndrome, vitamin D supplementation.16 In our case, the infant was not Sanjad-Sakati syndrome, Kearns-Sayre syndrome).5,20 Another breastfed, and he did receive the recommended dose of vitamin important etiology of hypoparathyroidism is an activating muta- D for infants. Moreover, the course of disease was prolonged with tion of the calcium-sensing receptor.21,22 Late hypocalcemia with persistent hypocalcemia. Usually, the normal calcium level can be high PTH levels is associated with pseudohypoparathyroidism or achieved within a mean period of 3 days.1,18 The causes of the secondary hyperparathyroidism induced by either the intake of patient's low 25-hydroxy-vitamin D level and for the recent find- phosphorus-rich milk or vitamin D deficiency. Iatrogenic causes in- ing of fatty liver are still under investigation. clude citrated blood products, diuretics, glucocorticoids, phosphate therapy, and phototherapy. A well-recognized risk factor for hypo- It also is important to recognize the high level of iPTH at pre- calcemia is hypomagnesemia. Hypomagnesemia can be a primary sentation. The combination of hypocalcemia and high PTH raises disorder or secondary to other diseases.23 Magnesium is necessary the suspicion of pseudohypoparathyroidism, specifically pseudo- for both PTH secretion and peripheral responsiveness to PTH. hypoparathyroidism type 1B, due to a lack of the typical features of Albright hereditary osteodystrophy. However, a diagnosis of In a recent study from Texas, the prevalence of moderate-to- pseudohypoparathyroidism should not be assigned in the setting of severe, late-onset neonatal hypocalcemia was 0.02%; it was more low vitamin D levels.27 In this case, the correction of 25-hydroxy- common in Hispanic and male infants and was readily managed vitamin D resulted in the normalization of the iPTH level. with therapy of limited duration (median duration of admission was 3 days).1 The treatment regimen included calcium and mag- Case 2 points out another important issue; despite very low 25- nesium supplements, low phosphorus formula, and supplementa- hydroxy-vitamin D levels, the 1,25-dihydroxyvitamin D level was tion with both the activated form of vitamin D (calcitriol) and within the reference range. The half-life of 1,25-dihydroxyvitamin its inactive parent compounds (vitamin D2 or D3). In a single D is short (4–6 hours), and the level may be reduced, normal, or ele- case report, recombinant PTH (subcutaneous 5 μg teriparatide) vated in cases of vitamin D deficiency, depending on the activity in a 17-day-old neonate successfully corrected hypocalcemia of 1α-hydroxylase that converts 25-hydroxy-vitamin D to 1,25- due to hypoparathyroidism.24 A decision tree management of dihydroxyvitamin D.6 In this patient with an elevated PTH level, neonatal hypocalcemia is illustrated in Figure 1. the 1α-hydroxylase was activated, resulting in normal levels of 1,25-dihydroxyvitamin D. Measuring the active form of vitamin D Case 1 is an example of neonatal hypocalcemia with seizures (1,25-dihydroxyvitamin D) is valuable in the evaluation of congenital due to maternal primary hyperparathyroidism. The mother was impaired vitamin D metabolism caused by mutations in the genes for clinically asymptomatic; however, recurrent blood test revealed 1α-hydroxylase, 25-hydroxylase, or the vitamin D receptor.28 serum hypercalcemia and hypophosphatemia as well as a high level of iPTH. In the fetus, maternal hyperparathyroidism results In case 3, the cause of the seizure episodes was DiGeorge syn- in high concentrations of serum calcium that acts to suppress the drome. The syndrome was first described by DiGeorge in 1965 as a parathyroid glands, resulting in a low fetal PTH concentration. syndrome of conotruncal cardiac anomalies, facial dysmorphism, hy- At birth, the neonate is suddenly deprived of this rich source of poplastic thymus, and hypocalcemia due to hypoplasia of the parathy- calcium. It is incapable of mobilizing calcium from bone owing roid glands.29 Since then, the syndrome has been described under to the low concentrations of PTH and high concentrations of cal- many different names, until the 1990s, when patients with different citonin. Acute neonatal hypocalcemia results in tetany and convul- phenotypes were found to share a deletion in band q11.2 of chromo- sions, usually at 5 to 14 days of age. Routine blood tests show some 22 as a common etiology.30,31 The syndrome has a wide pheno- hypocalcemia with an inappropriately low level of iPTH. Hypo- typic spectrum, with over 180 associated clinical manifestations magnesemia is also common. already described.32 Our patient presented some of these. She had mild facial dysmorphism, including a long face, high nasal root and The presentation of maternal asymptomatic primary hyperpara- bridge, and low-set ears. It is important to note that the “typical face” thyroidism through signs and symptoms in the neonate, although pre- of DiGeorge syndrome may not always be present, and facial viously documented anecdotally, is exceedingly rare.25,26 This case, dysmorphism may be subtle and thus sometimes overlooked.33 She like those previously documented, highlights the importance of care- also had a right aortic arch and chronic serous otitis media with hear- ful evaluation of neonatal hypoparathyroidism resulting in unco- ing loss, which may have been a consequence of palatal alterations.33 vering and treating hyperparathyroidism in the mother. In addition, it is interesting to note that the neonate was conceived through an In DiGeorge syndrome, hypocalcemia secondary to hypo- egg donor, and the patient's clinical manifestations after birth were parathyroidism manifests more often in the neonatal period, fre- solely dependent on the gestational environment in the surrogate. quently as seizures. During follow-up visits, our patient had hypercalciuria resulting in nephrocalcinosis. These manifestations Case 2 is an example of hypocalcemia due to neonatal vitamin D often arise as a consequence of excessive calcium replacement for deficiency. Vitamin D deficiency in a neonate has many etiologies and hypocalcemia, and they highlight the importance of measuring may be due to maternal vitamin D deficiency, exclusive prolonged calcium/creatinine ratio in the urine to avoid overtreatment. Fi- breast-feeding, or hereditary defects in vitamin D processing and activa- nally, the majority of individuals with DiGeorge syndrome have tion. Risk factors for maternal vitamin D deficiency include dark skin, behavior and learning problems34,35 as seen in our patient. Autism cultural modifications to diet or poor nutrition, minimal exposure to spectrum disorders are reported in up to 20% of patients.36,37 sunlight, and covered or veiled clothing. A recent study found that in- fants with hypocalcemic seizures and their mothers (study group) had In summary, we presented 3 cases of neonatal seizure sec- significantly lower 25-hydroxy-vitamin D levels than healthy breastfed ondary to hypocalcemia. Hypocalcemic seizures are now unusual, infants and their lactating mothers (control group).17 The mean level of and emergency medicine physicians should be familiar with this 25-hydroxy-vitamin D of the infants and mothers in the study group entity and the immediate need for treatment. Moreover, careful in- was 4.9 ± 4.6 and 6.5 ± 5.3, respectively, compared with 9.0 ± 4.6 vestigation of the specific etiology of the hypocalcemia is impor- and 9.1 ± 4.8 in the control group (P = 0.0001). tant to ensure better management and follow-up care. In our case report, the mother's 25-hydroxy-vitamin D level REFERENCES was 21 ng/mL (“desirable” reference range, >30 ng/mL). This value does not seem to be low enough to explain the very low level 1. 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Deletion 22q11: spectrum of associated disorders. Semin Pediatr Neurol. 2007;14:136–139. 19. Aggarwal R, Upadhyay M, Deorari AK, et al. Hypocalcemia in the newborn. Indian J Pediatr. 2001;68:973–975. 37. Kobrynski LJ, Sullivan KE. Velocardiofacial syndrome, DiGeorge syndrome: the chromosome 22q11.2 deletion syndromes. Lancet. 2007; 20. Al-Azem H, Khan AA. Hypoparathyroidism. Best Pract Res Clin 370:1443–1452. Endocrinol Metab. 2012;26:517–522. © 2015 Wolters Kluwer Health, Inc. All rights reserved. www.pec-online.com 201 Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. 188

‫פרויקט ח\"ץ ‪ -‬חינוך למצוינות רפואית‬ ‫במרכז הרפואי שיבא‪ ,‬תל השומר‬ ‫הרפואה | ‪2015‬‬ ‫מנחה‪ :‬פרופ' גדי סגל‬ ‫מנחה‪ :‬פרופ' ענת אחירון‬ ‫מנהל מחלקה פנימית ט'‪,‬‬ ‫מייסדת פרויקט ח\"ץ‪ ,‬מנהלת המרכז‬ ‫מנהל רשות ההוראה‬ ‫לטרשת נפוצה ואחראית הקתדרה‬ ‫למחלות אוטואימוניות אוניברסיטת ת\"א‬ ‫‪[email protected]‬‬ ‫‪[email protected]‬‬ ‫רותם אורבך‬ ‫אסף אחירון‬ ‫אונ' תל אביב‬ ‫אונ' תל אביב‬ ‫השתתפה כסטודנטית בפרויקט ח״ץ‬ ‫השתתף כסטודנט בפרויקט ח״ץ‬ ‫בין השנים ‪2007-2009‬‬ ‫בין השנים ‪2007-2009‬‬ ‫‪[email protected]‬‬ ‫‪[email protected]‬‬ ‫‪189‬‬

‫הרפואה • כרך ‪ • 154‬חוב' ‪ • 3‬מרץ ‪2015‬‬ ‫אקטואליה‬ ‫פרויקט ח\"ץ ‪ -‬חינוך למצוינות רפואית במרכז‬ ‫הרפואי שיבא‪ ,‬תל השומר‬ ‫אסף אחירון‪2,1‬‬ ‫לאורך ההיסטוריה‪ ,‬תגליות רבות ברפואה נעשו על ידי סטודנטים‪ Jay McLean .‬גילה את‬ ‫תקציר‪:‬‬ ‫רותם אורבך‪2,1‬‬ ‫ההפרין בהיותו סטודנט לרפואה בשנה השנייה; ל–‪ Paul Langerhans‬היו שתי תגליות‬ ‫חשובות במהלך לימודי הרפואה שלו‪ :‬הוא זיהה לראשונה תאים דנדריטיים בעור‬ ‫גד סגל‪3‬‬ ‫(הידועים עד היום כ–‪ )Langerhans cells‬ואפיין את ה–‪ Pancreatic islets‬בלבלב‪ ,‬שעד‬ ‫ענת אחירון‪3‬‬ ‫אז נחשב כבלוטת רוק; הסטודנט ‪ Charles Herbert Best‬היה שותף לגילוי האינסולין‬ ‫ולפרס נובל בהמשך‪ .‬גם גילוי ה–‪ Sino-atrial node‬וביצוע הרדמה באמצעות אתר הם‬ ‫‪1‬סטודנט ח\"ץ‪ ,‬מרכז רפואי שיבא‪ ,‬תל השומר‪ ,‬רמת גן‬ ‫‪2‬הפקולטה לרפואה סאקלר‪ ,‬אוניברסיטת תל אביב‬ ‫חלק מתגליות משמעותיות שנעשו על ידי סטודנטים לרפואה [‪.]1‬‬ ‫‪3‬מרכז רפואי שיבא‪ ,‬תל השומר‪ ,‬רמת גן‬ ‫פנדמיה; שפעת; חיסון; אתיקה‪.‬‬ ‫מילות מפתח‪:‬‬ ‫‪.Pandemic; Influenza; Vaccine; Ethics‬‬ ‫‪:KEY WORDS‬‬ ‫תרשים ‪:1‬‬ ‫הקדמה‬ ‫מספר הסטודנטים בפרויקט ח\"ץ לאורך זמן‬ ‫תרומתם האפשרית של סטודנטים צעירים‪ ,‬מחוננים ונחושים‬ ‫המסוגלים ליישם ידע וטכנולוגיות חדשניות והאפשרויות להישגיהם‬ ‫‪35‬‬ ‫במחקר מדעי קליני ובסיסי‪ ,‬עמדו בבסיס רעיון הקמת תוכנית ח\"ץ‬ ‫‪31 30 30‬‬ ‫(חוקר צעיר) במרכז הרפואי שיבא‪ .‬מטרת הפרויקט היא להכשיר‬ ‫למחקר רפואי סטודנטים כבר בשנות לימודיהם הראשונות על ידי‬ ‫‪25‬‬ ‫מס' הסטודנטים‬ ‫שילובם במחקרים קליניים בטווח נרחב של נושאים רפואיים‪.‬‬ ‫‪13 15‬‬ ‫‪20‬‬ ‫‪9 10‬‬ ‫‪15‬‬ ‫שיטות‬ ‫‪65‬‬ ‫‪10‬‬ ‫‪5‬‬ ‫פרויקט ח\"ץ הוקם בשנת ‪ 2007‬על ידי פרופסור ענת אחירון ובמסגרתו‬ ‫משתלבים במחקר במרכז הרפואי שיבא‪ ,‬תל השומר‪ ,‬סטודנטים‬ ‫‪2007 2008 2009 2010 2011 2012 2013 2014 0‬‬ ‫לרפואה מאוניברסיטת תל אביב‪ ,‬מהמסלולים הארבע והשש שנתי וכן‬ ‫ממסלול ניו יורק‪ .‬לאחר תהליך המיון וראיון הקבלה עם סגל הרופאים‬ ‫תרשים ‪:2‬‬ ‫הבכירים‪ ,‬הסטודנט מצוות למנחה בנושא מחקר ספציפי‪ .‬הסטודנטים‬ ‫‪150‬‬ ‫משתתפים במחקר באופן פעיל‪ ,‬לעיתים מראשית גיבוש הרעיון או‬ ‫חוקרי שיבא המשתתפים בפרויקט ח\"ץ על פי התמחות‬ ‫שאלת המחקר‪ ,‬במהלך המחקר‪ ,‬תוך כדי איסוף נתונים ועיבודם‪ ,‬ועד‬ ‫ריאות‬ ‫להצגת תוצאות המחקר בכנסים רפואיים ובפרסומים מדעיים‪.‬‬ ‫קרדיולוגיה‬ ‫הליך הלמידה במחקר כולל שרשרת של אירועים מבחירת נושא‬ ‫פתולוגיה‬ ‫מחקר ספציפי‪ ,‬סקירת הספרות העדכנית בנושא המחקר‪ ,‬הגשת‬ ‫פרמקולוגיה‬ ‫המחקר לאישור ועדת הלסינקי‪ ,‬בחירת קבוצות מחקר מתאימות‪,‬‬ ‫פנימית‬ ‫לימוד ותרגול סטטיסטי‪ ,‬לימוד מקצועי של טכנולוגיות וטכניקות‬ ‫פלסטיקה‬ ‫נדרשות‪ ,‬יישום השיטות הסטטיסטיות המתאימות לניתוח הנתונים‪,‬‬ ‫עיניים‬ ‫הכנת הרצאות והצגתן לשאר חברי הפרויקט‪ ,‬ולבסוף כתיבה מדעית‬ ‫אונקולוגיה‬ ‫ילדים‬ ‫ופרסום‪.‬‬ ‫גנטיקה‬ ‫אורתופדיה‬ ‫תוצאות‬ ‫דימות‬ ‫נירולוגיה‬ ‫מספר הסטודנטים המשתתפים בפרויקט ח\"ץ לאורך השנים מובא‬ ‫בתרשים ‪ .1‬מספר הסטודנטים עלה באופן הדרגתי עד ל–‪ 30‬משתתפים‬ ‫‪76 5 432 10‬‬ ‫בשנת ‪ .2014‬מספר חוקרי שיבא על פי התמחות המשתתפים בהדרכת‬ ‫סטודנטים מובא בתרשים ‪ ,2‬כאשר חלק מהחוקרים מנחים מספר‬ ‫השתתפו הסטודנטים מובאים בטבלה ‪ .1‬במהלך שבע שנות פעילות‪,‬‬ ‫סטודנטים‪ .‬פירוט נרחב של נושאי המחקר ניתן למצוא באתר פרויקט‬ ‫פירסמו הסטודנטים ‪ 20‬פרסומים מדעיים ‪ -‬הישג מרשים השווה‬ ‫ח\"ץ‪/‬סגן דיקאן שיבא בקישור‪http://www.medical-students. :‬‬ ‫‪ .tau.sheba.co.il/477‬פרסומי תוצאות המחקרים המדעיים שבהם‬ ‫בהיקפו למינוי מרצה בכיר בפקולטה לרפואה‪.‬‬ ‫‪190‬‬

‫אקטואליה‬ 2015 ‫ • מרץ‬3 '‫ • חוב‬154 ‫הרפואה • כרך‬ :1 ‫טבלה‬ ‫פרסומי הסטודנטים בפרויקט ח\"ץ‬ ‫המאמר המדעי‬ ‫שם‬ ‫הסטודנט שם המנחה‬ Prediction of acute multiple sclerosis relapses by transcription levels of peripheral blood cells. Gurevich M, Tuller T, Rubinstein ‫ד\"ר גורביץ מיכאל‬ ‫אורבך‬ 1 U, Or-Bach R, Achiron A. BMC Med Genomics. 2009 Jul 22;2:46. ‫רותם‬ Interleukin-12p40 in the spinal fluid as a biomarker for clinically isolated syndrome.Orbach R, Gurevich M, Achiron A. Mult Scler. ‫פרופ' אחירון ענת‬ ‫ אורבך‬2 2014 Jan;20(1):35-42. ‫רותם‬ Superior temporal gyrus thickness correlates with cognitive performance in multiple sclerosis. Achiron A, Chapman J, Tal S, ‫ אחירון פרופ' צ'פמן יואב‬3 Bercovich E, Gil H, Achiron A. Brain Struct Funct. 2013 Jul;218(4):943-50. ‫אסף‬ Thrombin regulation of synaptic plasticity: implications for physiology and pathology. Maggio N, Itsekson Z, Dominissini D, Blatt ‫פרופ' צ'פמן יואב‬ ‫איזקסון‬ 4 I, Amariglio N, Rechavi G, Tanne D, Chapman J. Exp Neurol. 2013 Sep;247:595-604. ‫זאב‬ Circulating angiogenic factors in monochorionic twin pregnancies complicated by twin-to-twin transfusion syndrome and ‫פרופ' ליפיץ שלמה‬ ‫בן מאיר‬ 5 selective intrauterine growth restriction. Yinon Y, Ben Meir E, Berezowsky A, Weisz B, Schiff E, Mazaki-Tovi S, Lipitz S. Am J ‫ד\"ר ינון יואב‬ ‫אלעד‬ Obstet Gynecol. 2014 Feb;210(2):141.e1-7. Late diagnosis of fetal central nervous system anomalies following a normal second trimester anatomy scan. Yinon Y, Katorza E, ‫פרופ' ליפיץ שלמה‬ ‫בן מאיר‬ 6 Nassie DI, Ben-Meir E, Gindes L, Hoffmann C, Lipitz S, Achiron R, Weisz B. Prenat Diagn. 2013 Oct;33(10):929-34. ‫ד\"ר ינון יואב‬ ‫אלעד‬ Ber R , Bar-Yosef O, Hoffmann C, Shashar D, Achiron R, Katorza E .Normal Fetal Posterior Fossa in MR Imaging: New Biometric ‫ בר רועי ד\"ר קטורזה אלדד‬7 Data and Possible Clinical Significance.AJNR Am J Neuroradiol. 2015 Feb 5 Regional apparent diffusion coefficient values in 3rd trimester fetal brain. Hoffmann C, Weisz B, Lipitz S, Yaniv G, Katorza E, ‫ד\"ר הופמן חן‬ ‫ברגמן‬ 8 Bergman D, Biegon A. Neuroradiology. 2014 Apr 20. ‫דפי‬ Pituitary function in children following infectious diseases of the central nervous system. Levy-Shraga Y1, Gazit I, Modan-Moses ‫ד\"ר חמיאל אורית‬ ‫גזית‬ 9 D, Pinhas-Hamiel O. Pituitary. 2014 Apr;17(2):118-24. doi: 10.1007/s11102-013-0476-2. ‫ענבל‬ The Many Etiologies of Neonatal Hypocalcemic Seizures. Yael Levy- Levy-Shraga Y, Dallalzadeh K, Stern K, Paret G, Pinhas- ‫ד\"ר לוי יעל‬ ‫דללזדה‬ 10 Hamiel O. Pediatr Emerg Care. 2015 Mar;31(3):197-201. ‫קרן‬ Immunization with hepatitis B vaccine accelerates SLE-like disease in a murine model. Agmon-Levin N, Arango MT, Kivity S, ‫ וולקוב ד\"ר אגמון–לוין ננסי‬11 Katzav A, Gilburd B, Blank M, Tomer N, Volkov A, Barshack I, Chapman J, Shoenfeld Y. J Autoimmun. 2014 Jul 16. pii: S0896- ‫אילן‬ 8411(14)00103-6. doi: 10.1016/j.jaut.2014.06.006. Anogenital distance in male and female fetuses at 20 to 35weeks of gestation: centile charts and reference ranges. Gilboa Y, ‫ד\"ר גלבוע ינון‬ ‫כהן–פז‬ 12 Kivilevitch Z, Oren M, Cohen YP, Katorza E, Achiron R. Prenat Diagn. 2014 May 12. doi: 10.1002/pd.4399. ‫יסכה‬ Phosphorylcholine-tuftsin compound prevents development of dextran-sulfate-sodium-induced murine colitis.Dana Ben-Ami, ‫פרופ' בלנק מירי‬ ‫לחניש‬ 13 Tomer Bashi, Jordan Lachnish, Mati Fridkin, Giorgia Bizzaro, Iris Barshak, Miri Blank, Yehuda Shoenfeld. CRAI 2014, in press. ‫ירדן‬ Effective crizotinib schedule for brain metastases in ALK rearrangement metastatic non-small-cell lung cancer. Peled N, Zach L, ‫ד\"ר פלד ניר‬ ‫לירן‬ 14 Liran O, Ilouze M, Bunn PA Jr, Hirsch FR. J Thorac Oncol. 2013;8(12):e112-3. ‫אורי‬ Pupillometer-based objective chromatic perimetry in normal eyes and patients with retinal photoreceptor dystrophies. Skaat A, Sher I, ‫ד\"ר רוטנשטרייך‬ ‫מהאג'נה‬ 15 Kolker A, Elyasiv S, Rosenfeld E, Mhajna M, Melamed S, Belkin M, Rotenstreich Y. Invest Ophthalmol Vis Sci. 2013 Apr 17;54(4):2761-70. ‫יגאל‬ ‫מוחמד‬ Optimizing multiple sclerosis diagnosis: gene expression and genomic association. Gurevi M, Miron G, Achiron A. Annals of ‫ד\"ר גורביץ מיכאל‬ ‫מירון‬ 16 Clinical and Translational Neurology. DOI:10.1002/acn3.174 ‫גדי‬ Potential drug interactions in travelers with chronic illnesses: A large retrospective cohort study. Stienlauf S, Meltzer E, Kurnik D, Leshem ‫פרופ' שוורץ אלי‬ ‫סטרלצין‬ 17 E, Kopel E, Streltsin B, Schwartz E. Travel Med Infect Dis. 2014 May 5. pii: S1477-8939(14)00082-9. doi: 10.1016/j.tmaid.2014.04.008 ‫ד\"ר שטינלאוף‬ ‫ביאנקה‬ ‫שמואל‬ 16/6-idiotype expressing antibodies induce brain inflammation and cognitive impairment in mice: the mosaic of central nervous ‫פרופ' שינפלד‬ ‫רבי‬ 18 system involvement in lupus. Kivity S, Katzav A, Arango MT, Landau-Rabi M, Zafrir Y, Agmon-Levin N, Blank M, Anaya JM, Mozes ‫יהודה‬ ‫מורן‬ E, Chapman J, Shoenfeld Y. BMC Med. 2013 Apr 4;11:90. doi: 10.1186/1741-7015-11-90. Mitochondrial induction as a potential radio-sensitizer in lung cancer cells - a short report. Shavit R, Ilouze M, Feinberg T, ‫פרופ' ניר פלד‬ ‫רונן‬ 19 Lawrence YR, Tzur Y, Peled N. Cell Oncol (Dordr). 2015 Jan 7. [Epub ahead of print] ‫שביט‬ Incomplete response to colchicine in M694V homozygote FMF patients. Lidar M, Yonath H, Shechter N, Sikron F, Sadetzki S, ‫ שחטר ד\"ר יונת חגית‬20 Langevitz P, Livneh A, Pras E. Autoimmun Rev. 2012 Nov;12(1):72-6. ‫נעמה‬ ,‫ מנהל המרכז הרפואי שיבא‬,‫ ברצוננו להודות לפרופ' זאב רוטשטיין‬:‫תודות‬ ‫לסיכום‬ ‫על תמיכתו לאורך כל הדרך ולגב' יפית רוט על ארגונה המסור להצלחת‬ .‫ לגיל הררי ולליעד שקל על הייעוץ הסטטיסטי‬,‫הפרויקט‬ ,‫ המצוי עדיין בתהליך צמיחה וגדילה‬,‫פרויקט ח\"ץ הוא פרויקט ייחודי‬ ‫ביבליוגרפיה‬ •‫ואשר נותן כבר את פירותיו בבניית העתודה הרפואית החוקרת‬ .‫במדינת ישראל‬ 1. Stringer MD & Ahmadi O, Famous discoveries by medical students. ‫ אסף אחירון‬:‫מחבר מכותב‬ [email protected] :‫דוא\"ל‬ 151 ANZ J Surg, 2009;79:901-8. 191



‫‪Optimizing multiple sclerosis diagnosis:‬‬ ‫‪gene expression and genomic association‬‬ ‫‪Annuals of clinical and translational neurology | 2015‬‬ ‫מנחה‪ :‬דר' מיכאל גורביץ‬ ‫מנחה‪ :‬פרופ' ענת אחירון‬ ‫מנהל המעבדה הנוירואימונולוגית‪,‬‬ ‫מייסדת פרויקט ח\"ץ‪ ,‬מנהלת המרכז‬ ‫המרכז לטרשת נפוצה‬ ‫לטרשת נפוצה ואחראית הקתדרה‬ ‫למחלות אוטואימוניות אוניברסיטת ת\"א‬ ‫‪[email protected]‬‬ ‫‪[email protected]‬‬ ‫גדי מירון‬ ‫אונ' תל אביב‬ ‫השתתף כסטודנט בפרויקט ח״ץ‬ ‫בין השנים ‪2012-2014‬‬ ‫‪[email protected]‬‬ ‫‪193‬‬

RESEARCH ARTICLE Optimizing multiple sclerosis diagnosis: gene expression and genomic association Michael Gurevich1, Gadi Miron1 & Anat Achiron1,2 1Multiple Sclerosis Center, Sheba Medical Center, Ramat-Gan, Israel 2Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel Correspondence Abstract Michael Gurevich, Multiple Sclerosis Center, Sheba Medical Center, Tel-Hashomer, Ramat- Objective: The diagnosis of multiple sclerosis (MS) at disease onset is some- Gan 52621, Israel. Tel: 97235305785; Fax: times masqueraded by other diagnostic options resembling MS clinically or 97235348186; E-mail: gurevich@sheba. radiologically (NonMS). In the present study we utilized findings of large-scale health.gov.il Genome-Wide Association Studies (GWAS) to develop a blood gene expres- sion-based classification tool to assist in diagnosis during the first demyelinating Funding Information event. Methods: We have merged knowledge of 110 MS susceptibility genes This study has received no public or private gained from MS GWAS studies together with our experimental results of differ- funding. ential blood gene expression profiling between 80 MS and 31 NonMS patients. Multiple classification algorithms were applied to this cohort to construct a Received: 20 November 2014; Revised: 23 diagnostic classifier that correctly distinguished between MS and NonMS December 2014; Accepted: 23 December patients. Accuracy of the classifier was tested on an additional independent 2014 group of 146 patients including 121 MS and 25 NonMS patients. Results: We have constructed a 42 gene-transcript expression-based MS diagnostic classifier. Annals of Clinical and Translational The overall accuracy of the classifier, as tested on an independent patient Neurology 2015; 2(3): 271–277 population consisting of diagnostically challenging cases including NonMS patients with positive MRI findings, achieved a correct classification rate of doi: 10.1002/acn3.174 76.0 Æ 3.5%. Interpretation: The presented diagnostic classification tool com- plements the existing diagnostic McDonald criteria by assisting in the accurate exclusion of other neurological diseases at presentation of the first demyelinat- ing event suggestive of MS. Introduction only have immunological and inflammatory properties, but are also specifically associated with MS pathogenesis. The diagnosis of multiple sclerosis (MS) is a challenging Large-scale genome-wide association studies (GWAS) procedure and is currently based on the 2010 McDonald examining single-nucleotide polymorphisms (SNP’s) criteria requiring evidence of disease dissemination in time encompassing thousands of patients have been conducted (DIT), dissemination in space (DIS) and “no better expla- and have established 110 MS-related SNP’s,8–11 that con- nation” for symptoms.1 At first demyelinating symptom- tribute to MS susceptibility and pathophysiology. It has atology, clinical, imaging and laboratory findings may be been previously demonstrated that SNPs could affect similar between MS and other diseases that mimic MS expression of nearby genes.12 Therefore, we have merged (NonMS) including infectious, neoplastic, metabolic, our findings of gene expression of MS susceptible loci vascular or idiopathic inflammatory demyelinating dis- genes with other highly differentially expressed genes eases.2–4 Therefore, a need exists to find accurate biomar- (DEGs) between MS and NonMS patients to develop a kers and to develop a tool that can assist in the exclusion of blood gene expression-based diagnostic classification tool. NonMS diseases. This multi-gene classifier was tested on an independent cohort of patients during the first neurological event High throughput technology measuring simultaneous suggestive of MS demonstrating high accuracy and there- expression of thousands of genes or proteins has been fore could improve the diagnostic process in challenging shown to be potentially useful for diagnosis of MS.5–7 cases. However, it is challenging to find biomarkers that not ª 2015 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association. 271 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. 194

Optimizing Multiple Sclerosis Diagnosis G. Michael et al. Methods Patients We analyzed blood samples of patients selected according to the following criteria: (1) Age 18–60 years; (2) Free of steroid treatment for at least 30 days; (3) Patients with the first clinical demyelinating event suggestive of MS that upon presentation fulfilled DIT or DIS according to 2010 McDonald’s criteria. After follow-up period of 5 years these patients were further divided into two groups: (a) patients that converted to relapsing remitting MS (RRMS) (Clinically Isolated Syndromes, CIS group); (b) patients that did not convert to MS and after extensive work-up a better explanation for the clinical and imaging findings was established (NonMS group). (4) Patients that at the time of sampling fulfilled McDonald 2010 criteria for RRMS and served for classifier training and testing. Standard protocol approvals and patients Figure 1. Flowchart of study design. Samples from 257 patients consent including 137 patients at first demyelinating event and 120 RRMS patients were subjected to gene expression microarray analysis and The study was approved by Sheba Medical Center Institu- randomly divided into a training set (n = 111) and test set (147). tional Review and Ethical Board and all patients gave training set was used for diagnostic classifier generation and then written informed consent. Demographic and clinical data classifier performance was validated on independent test set. were retrieved from Sheba Multiple Sclerosis Center com- Resampling (n = 43) was done to demonstrate classifier consistency. puterized database. DNase digestion step. RNA quality was determined by Study design BioRad Experion (Hercules, California, USA) automatic electrophoresis station. cDNA was synthesized from 3 lg Patient data set (n = 257) was divided into two sub- total RNA using the One-Cycle cDNA Synthesis Kit, and groups: training set (n = 111) and test set (n = 146). The in vitro transcription was performed with the GeneChip training set was used to construct blood gene expression- IVT Labeling Kit (Affymetrix, Inc., Santa Clara, CA). The based classifier, while the independent test set allowed for biotin-labeled IVT-RNA was hybridized to HGU133A-2 subsequent testing of the classifier performance. Multiple arrays containing ~22,000 gene transcripts corresponding classification algorithms implemented in Partek software to 14,500 well-annotated human genes, washed in a Gene- (St. Louis, Missouri, USA) were applied to the training Chip Fluidics Station 450 and scanned on GeneArray-TM set subgroups to construct classifiers that can correctly scanner (G2500A; Hewlett Packard Palo Alto, California, distinguish between MS and NonMS patients. The classi- USA) according to standard Affymetrix Inc. protocol. fier with the best performance on the training set was validated on the test set. Data pretreatment, normalization, and statistical analysis An additional internal cross-validation was performed by testing the classifier consistency on a subgroup of Following scanning of the arrays, the microarray raw data patients that were resampled within 0.8 Æ 0.2 years from were initially normalized by R Bioconductor Packages13 the initial blood sampling. The study flow chart is dem- as follows: (1) all arrays were normalized serially using a onstrated in Figure 1. single sample microarray normalization approach designed for personal medicine workflows (SCAN Nor- Microarray preparation malization)14; (2) an empirical bayes approach was used to address batch effect in the data as implemented in Total RNA from patients frozen peripheral blood mono- Combat SVA package.15,16 nuclear cells (PBMC) was extracted using Trizol (Invitro- gen, Carlsbad, California, USA) and Phase-Look-Gel columns (Eppendorf, Hamburg, Germany) including a 272 ª 2015 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association. 195

G. Michael et al. Optimizing Multiple Sclerosis Diagnosis Partek Genomics Software (www.partek.com) was used Demographic and clinical variables of the study for data analysis. All genes related to clinical and demo- patients in relation to experimental training set and test graphical confounders such as age, gender, immunomodu- set groups are presented in Table 1. latory treatment with P < 0.01 were excluded from further analysis. Next, to select candidate biomarkers for the classi- MS diagnostic classifier generation fier we performed comparison gene expression analysis between MS and NonMS patients in the training set. Genes The diagnostic classifier was generated based on the train- selected were comprised of: (1) DEGs within the entire ing set of samples from 111 patients including 80 MS and transcriptome with P value <0.05 after Bonferroni correc- 31 NonMS. Gene expression analysis performed on these tion for multiple comparisons and (2) DEGs with P groups identified 29 DEG’s with P < 0.05 after Bonferroni value<0.05 from 207 gene transcripts corresponding to 110 correction and 49 GWAS DEGs, resulting in 78 candidate loci found to be associated with MS in published GWAS genes for generating the diagnostic classifier. These 78 (GWAS DEG).9,10,17,18 These genes were then used to build candidate biomarkers were used to construct and opti- a support vector machine (SVM) classifier. The SVM was mize classification accuracy of various SVMs. The most configured as cost based, with costs varied from 1 to 1001 accurate SVM classifier used expression intensity of 42 in intervals of 100. The tolerance (termination criterion) gene – transcripts (Table S1) to correctly classify patients was set at 0.001. The kernel for the SVM was a polynomial in the training set with a total correct rate of 94.6%, hav- function, with gamma equal to the inverse of the number of ing a sensitivity of 95.0 Æ 2.4% and a specificity of evaluated probes. The optimal SVM was derived by shrink- 94.0 Æ 4.4%. Within the 42 transcripts chosen for the ing centroids and 10-fold cross-validation. Principal Com- classifier, 18 were based on SNP’s reported in the GWAS ponent Analysis (PCA) was used for visualization of results. studies. PCA of the training set based on these 42 tran- scripts is shown in Figure 2. Functional analysis of genes included in diagnostic classifier Verification: classifier testing on the independent test set Biological functional analysis of the classifier genes was performed by Ingenuity Pathway Analysis (IPA) software The MS diagnostic classifier performance was verified on (www.ingenuity.com) that links gene products with bio- an independent test set, consisting of samples obtained logical processes, molecular function and cellular compo- from CIS (n = 58), RRMS (n = 63) and NonMS (n = 25) nents. Right-tailed Fisher’s exact test was used to calculate patients. First, the ability of the diagnostic classifier was a P value determining the probability that each biological tested in the group of RRMS patients and NonMS function assigned to gene data set is not due to random patients, demonstrating overall correct classification rate chance. The P values obtained from Fisher’s analysis were of 77.3 Æ 4.5%, sensitivity of 78.0 Æ 5.2% and specificity applied for Benjamini-Hochberg False Discovery Rate 76.0 Æ 8.5% for the diagnosis of MS patients. Although (FDR) multiple testing corrections to keep the overall RRMS patients already have an established diagnosis, this error rate at P < 0.05. step was done to prove classifier ability to identify MS specific expression signature. Next, to assess clinical appli- Results cability, the classifier was applied to diagnostically rele- vant cohort of CIS and NonMS patients at the time of Patients Table 1. Demographical and clinical characteristics of patients. Blood samples were obtained from 137 patients that presented with symptomatology suggestive of MS at Group N Age average F (M) EDSS disease onset; 81 were diagnosed with CIS and 56 were diagnosed as NonMS patients. The CIS group included 50 Training set CIS 23 31.6 Æ 1.4* 16 (7) 1.4 Æ 0.3 females and 31 males, mean Æ SE age 31.9 Æ 1.2 years, the NonMS group included 43 females and 13 males, RRMS 57 36.0 Æ 1.4* 36 (12) 2.1 Æ 0.2 mean Æ SE age 41.9 Æ 1.5 years. NonMS 31 41.6 Æ 2.3 24 (7) NR The group of 120 RRMS patients that served for the classifier training and testing included 80 females and 40 Total 111 36.6 Æ 0.1 76 (35) NR males, mean Æ SE age 37.3 Æ 1.0 years, disease duration 5.6 Æ 0.5 years and Expanded Disability Status Scale Test set CIS 58 32.2 Æ 1.4* 23 (35) 1.4 Æ 0.1 (EDSS) 2.1 Æ 0.1. RRMS 63 38.4 Æ 1.4* 43 (20) 2.2 Æ 0.2 NonMS 25 42.3 Æ 1.7 19 (6) NR Total 146 36.6 Æ 0.9 97 (49) NR RRMS, relapsing remitting multiple sclerosis; NR, not relevant. *P < 0.05 as compare to NonMS group. ª 2015 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association. 273 196

Optimizing Multiple Sclerosis Diagnosis G. Michael et al. Figure 2. Principal component analysis (PCA) based on 42 gene- set the consistency of results was 85.0% and in the train- transcripts of the diagnostic classifier. This difference between MS ing set the consistency was 78.0%. and NonMS patients from training set is presented. Each dot represents patient sample principal components derived from As could be seen from Table 1, in the training and test expression of 42 diagnostic classifier gene-transcripts. The distance set the age of MS and NonMS patients was significantly between any pair of points is related to the similarity between the different. To analyze the effect of age on classifier perfor- two observations in high-dimensional (3D) space. Blue dots represent mance we examined the 42 gene-transcripts classifier per- NonMS patients, Red dots represent MS patients. formance in age-matched subgroups. This analysis demonstrated that, overall classifier ability to diagnose the first demyelinating event (n = 83). In this cohort the MS and NonMS patients was not affected by age. This is classifier demonstrated an overall accuracy of 74.7 Æ shown by the similar range of correct classification of 4.8%, sensitivity 74.0 Æ 5.7%, and specificity 76.0 Æ 81.4 Æ 7.5%, 72.2 Æ 7.5%, 72.7 Æ 6.7%, and 78.4 Æ 8.5% for the diagnosis of MS. The overall accuracy of the 6.7% in the respective age subgroups <25, 25–35, 35–45, classifier on the entire test set of independent patients and >45 years. (n = 146) was 76.0 Æ 3.5%, Table 2. To assess the predictive contribution added by GWAS Further analysis of classifier performance showed DEGs to DEG’s we constructed two additional classifiers, dependence of the classification accuracy on the initial the first based solely on GWAS DEGs genes and another clinical disability score of patients. Thus, including only based only on DEG’s. Both classifiers performed inferiorly patients with abnormal neurological findings (EDSS > 0) to the 42 gene-transcript classifier, with the GWAS DEGs at the time of sampling, improved the classifier sensitivity classifier achieving an accuracy rate of 59.0 Æ 4.6% with to 78.0 Æ 5.2% in the CIS patient group, and to sensitivity 55.0 Æ 5.2% and specificity 72.0 Æ 9.0% and 80.4 Æ 4.0% overall (Table 2). the only DEG’s classifier demonstrating an accuracy of 76.0 Æ 3.9% with sensitivity of 83.0 Æ 3.9% but low level An additional internal cross-validation test of the classi- of specificity 53.0 Æ 10.0%. fier validity was done by analysis of repeated samples. Consistency of the classifier to reproduce the same classi- Functional analysis of diagnostic classifier fication was tested in 43 patient samples retaken after an genes average time of 0.8 Æ 0.2 years from the initial sampling. Twenty samples were retested from the test set group, Functional analysis of the classifier genes showed enrich- and 23 samples were retested from the training set group. ment of genes associated with different mechanisms of Consistency of the results was overall 81.4%. In the test cell movement and migration (P value 3.21E-03 to 1.54E- 05), immune cell trafficking including lymphocytes migra- tion (P = 1.54E-05), cell movement of T lymphocytes (P = 6.79E-05), transmigration of T lymphocytes (P = 1.97E-04) and adhesion of immune cells (P = 1.49E-03), Table S2. These findings were further consistent with gene expression network analysis in which the classifier genes were shown to organize in four functional networks, including cell-mediated immune response and immune cell trafficking, similarly to the results of the enrichment analysis, Table S3. Additionally, the classifier genes were found to be upstream regulators of other molecules, including CXCR4 and JAK2 regulating CDH1. Further- more, these regulators were related not only to Table 2. Summary of MS diagnostic classifier performance. Total accuracy Sensitivity Specificity Groups compared 74.7 Æ 4.8% 74.0 Æ 5.7% 76.0 Æ 8.5% 77.1 Æ 5.0% 78.0 Æ 6.2% 76.0 Æ 8.5% Test set CIS (n = 58) vs. NonMS (n = 25) 77.3 Æ 4.5% 78.0 Æ 5.2% 76.0 Æ 8.5% Test set CIS EDSS > 0 (n = 45) vs. NonMS (n = 25) 76.0 Æ 3.5% 76.0 Æ 3.9% 76.0 Æ 8.5% Test set RRMS (n = 63) vs. NonMS (n = 25) 78.9 Æ 3.6% 80.0 Æ 4.0% 76.0 Æ 8.5% Test set all MS (n = 121) vs. NonMS (n = 25) Test set all MS EDSS > 0 (n = 103) vs. NonMS (n = 25) RRMS, relapsing remitting multiple sclerosis. 274 ª 2015 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association. 197

G. Michael et al. Optimizing Multiple Sclerosis Diagnosis downstream molecules, but they also could be intercon- affecting splicing patterns, altering proteins by changing nected between themselves, Figure 3. single amino acids and modulating micro-RNA-binding sites activity.12,19 Although examining the specific mecha- Discussion nism by which each SNP affects its corresponding gene expression is beyond the scope of this study, overall we In this study, we present an innovative approach whereby have shown significant differential expression of 49 MS we developed a blood gene expression-based diagnostic susceptible loci associated transcripts, from which 18 were tool proved to be useful for excluding MS mimicking included in our diagnostic classifier. diseases in the early stage of the diagnostic process. For this purpose we have merged knowledge of MS susceptibility Currently, the McDonald criteria used for MS diagnosis genes gained from MS GWAS studies together with our have specificity over 90% with sensitivity of 77% and accu- experimental results of differential gene expression profil- racy of 86%.20–23 Notably, these high accuracy levels were ing between MS and NonMS patients. This multimodal achieved in multicenter trials conducted in tertiary MS cen- approach allowed us to utilize data acquired from very ters, after exclusion of patients with other neurological dis- large scale studies to focus on a narrow list of candidate bi- eases.1,22,24 Therefore, the relevance of the high accuracy of omarkers that contribute to the pathological mechanisms McDonald criteria has been questioned with regard to every- operating in MS. While certain genetic associations have day clinical practice in which NonMS patients are not been consistently shown to contribute to the etiology of excluded prior to application of the criteria.22 A study exam- MS,11 the link between genome variability and disease phe- ining the diagnostic performance of DIS criteria in such a notypes still remain poorly understood. To the best of our cohort of difficult cases mimicking MS, showed that the sen- knowledge our study is the first to demonstrate the associa- sitivity of the diagnosis decreased to 64%.22 tion between genomic variability and differential gene expression phenotypes of MS and other neurological dis- Our diagnostic classifier was able to successfully discrim- eases that mimic MS. SNP’s may influence gene expression inate between CIS converting to MS and NonMS patients in several ways, including modulating transcription factor- during the early disease stage with sensitivity of 74.0% and binding sites, changing amino sequence of stop codons, specificity of 76.0%, therefore having potential use for clini- cal practice. This diagnostic classifier may assist to reach the Figure 3. Functional regulatory network of classifier genes. Classifier correct diagnosis in patients with suspected MS at an early gene network reconstructed based on literature-known relationships stage. Importantly, the accuracy rate of the classifier was according to IPA software database. Each node in the regulation tree tested on population that consisted of diagnostically chal- represents a regulating gene, arrows indicate literature confirmed lenging cases of NonMS patients having positive findings regulatory interactions. Over-expressed genes are depicted in red, on MRI and fulfilling McDonald criteria for DIT or DIS. down-expressed in green. Furthermore, the EDSS-dependent improvement of the classifier performance to reach a sensitivity of 78.0%, sug- gests that the expression of genes involved correlate with patients’ neurological findings. This suggests that genes included in the classifier represent disease activity profile rather than the constitutive gene expression profile of MS disease as we have previously reported.25,26 Functional enrichment analysis of the genes included in the classifier identified genes and functions with signifi- cant role in MS pathophysiology. The most significantly enriched functions are related to immune cell trafficking and adhesion. Specifically, the regulatory network includ- ing CXCR4, a master regulator having a known role as a stimulator of T- and B-cell proliferation and migration through the endothelium.27–30 CXCR4 connects to the downstream CD6 gene encoding the SRCR domain and binding site for activated cell adhesion molecules, and further continues the process of T-cell activation. It is of note that both CXCR4 and CD6 are associated with MS susceptibility loci. This process involving immune cells activation, proliferation, and adhesion appears to play a central role in the classifier network. Two additional important genes in the network include MALT1 and ª 2015 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association. 275 198